Methods for treating disease using inhibitors of bone morphogenetic protein 6 (bmp6)

ABSTRACT

The present invention relates methods of treatment using BMP6 antagonists.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 23, 2017, isnamed PAT057354-WO-PCT_SL.txt and is 82,879 bytes in size.

INTRODUCTION

The present invention relates to methods of treating anemia usinginhibitors of bone morphogenetic protein 6 (BMP6).

BACKGROUND OF THE INVENTION

Anemia is prevalent in patients with chronic kidney disease (CKD) and isassociated with lower quality of life and higher risk of adverseoutcomes, including cardiovascular disease and death. Several modes ofanemia management in patients with CKD involve the use oferythropoiesis-stimulating agents (ESA), supplemental oral andintravenous iron and blood transfusions. However, many patients do notrespond adequately to these treatments or require higher doses of ESAand/or iron. High doses of iron may also cause toxicity associated withgeneration of oxygen radicals and allergic reactions. These treatmentsmay lack efficacy because they do not fully address the underlying causeof the anemia, i.e., impaired iron absorption and iron mobilization frombody stores.

Attempts to manage erythropoietin resistance are currently performed bythe co-administration of high dose parenteral iron. However, most ironfrom intravenous preparations is first processed by macrophages, and itsutilization for erythropoiesis is dependent on ferroportin-mediated ironexport.

In many anemia patients, ferroportin-mediated iron export is suppressedby high levels of hepcidin. Additional evidence suggests that increasedlevels of hepcidin correlate with poor ESA responsiveness inhemodialysis. Hepcidin-lowering agents may therefore be an effectivestrategy for ameliorating ESA-refractory anemia in this patientpopulation and in other forms of anemia of chronic disease (ACD)characterized by iron restriction.

Therefore, methods that decrease circulating hepcidin levels shouldenhance iron absorption, facilitate release of sequestered iron, andpromote erythropoiesis in ESA-refractory anemia present in chronickidney disease patients.

Despite current treatment options for treating diseases and disordersassociated anemia, there remains a need for improved method s oftreatment of anemia which are effective and well-tolerated.

SUMMARY OF THE INVENTION

Ferritin is an intracellular protein that is indicative of stored iron.Ferritin levels can be used as an indirect marker of the total amount ofiron stored in the body. Without being bound by theory, it is believedthat inhibitors of BMP6 may result the release of stored iron fromcells, for example, from macrophages and/or enterocytes. Again, withoutbeing bound by theory, the present invention is based in part on thediscovery that inhibitors of BMP6 may be effective in treatingconditions associated with sequestered iron, e.g., anemia, in patientswith high ferritin (e.g., pretreatment ferritin levels greater than 500e.g., serum ferritin, levels, and hence, high stored iron.

In a first aspect, the invention pertains to a method of selectively:

-   a. inhibiting BMP6;-   b. increasing serum iron levels, transferrin saturation (TAST),    reticulocyte hemoglobin content (CHr), reticulocyte count, red blood    cell count, hemoglobin, or hematocrit;-   c. reducing the activity or level of Hepcidin;-   d. treating anemia; or-   e. increasing or maintaining hemoglobin level;-   in a patient in need thereof, that includes selectively    administering a therapeutically effective amount of a BMP6    antagonist to the patient on the basis of a biological sample from    the patient having a ferritin level of ≤2000 ng/mL. In embodiments,    the ferritin level is ≥500 ng/mL.

In another aspect, the invention pertains to a method of treating apatient having anemia with a BMP6 antagonist, including selectivelyadministering a therapeutically effective amount of a BMP6 antagonist tothe patient on the basis of a biological sample from the patient havinga ferritin level of ≤2000 ng/mL. In embodiments, the ferritin level is≥500 ng/mL.

In another aspect, the invention pertains to a method of selectivelytreating a patient having anemia with a BMP6 antagonist, including:

-   a) assaying a biological sample from the patient for ferritin level;    and-   b) thereafter, selectively administering to the patient a    therapeutically effective amount of a BMP9 antagonist, wherein the    ferritin level is ≤2000 ng/mL. In embodiments, the ferritin level is    ≥500 ng/mL.

In another aspect, the invention pertains to a method of selectivelytreating a patient having anemia with a BMP6 antagonist, including:

-   a) assaying a biological sample from the patient for ferritin level;-   b) thereafter, selecting the patient for treatment with the BMP6    antagonist on the basis of the biological sample from the patient    having a ferritin level ≤2000 ng/mL; and-   c) thereafter, administering a therapeutically effective amount of a    BMP9 antagonist to the patient. In embodiments, the ferritin level    is ≥500 ng/mL.

In another aspect, the invention pertains to a method of selectively:

-   a. inhibiting BMP6;-   b. increasing serum iron levels, transferrin saturation (TAST),    reticulocyte hemoglobin content (CHr), reticulocyte count, red blood    cell count, hemoglobin, or hematocrit;-   c. reducing the activity or level of Hepcidin;-   d. treating anemia; or-   e. increasing or maintaining hemoglobin level;-   in a patient in need thereof, that includes selectively    administering a therapeutically effective amount of a BMP6    antagonist to the patient on the basis of a biological sample from    the patient having a ferritin level of ≥500 ng/mL.

In another aspect, the invention pertains to a method of treating apatient having anemia with a BMP6 antagonist, including selectivelyadministering a therapeutically effective amount of a BMP6 antagonist tothe patient on the basis of a biological sample from the patient havinga ferritin level of ≥500 ng/mL.

In another aspect, the invention pertains to a method of selectivelytreating a patient having anemia with a BMP6 antagonist, including:

-   a) assaying a biological sample from the patient for ferritin level;    and-   b) thereafter, selectively administering to the patient a    therapeutically effective amount of a BMP9 antagonist, wherein the    ferritin level is 500 ng/mL.

In another aspect, the invention pertains to a method of selectivelytreating a patient having anemia with a BMP6 antagonist, including:

-   a) assaying a biological sample from the patient for ferritin level;-   b) thereafter, selecting the patient for treatment with the BMP6    antagonist on the basis of the biological sample from the patient    having a ferritin level 500 ng/mL; and-   c) thereafter, administering a therapeutically effective amount of a    BMP9 antagonist to the patient.

In embodiments, including in any of the aforementioned aspects, theferritin level is ferritin protein level.

In embodiments, including in any of the aforementioned aspects, the stepof assaying includes a technique selected from the group consisting ofan immunoassay, immunohistochemistry, ELISA, flow cytometry, Westernblot, HPLC, and mass spectrometry.

In another aspect, the invention pertains to a BMP6 anagonist for use intreating a patient having anemia, characterized in that atherapeutically effective amount of the BMP6 antagonist is to beadministered to the patient on the basis of a biological sample from thepatient having a ferritin level of 2000 ng/mL. In embodiments, theferritin level is ≥500 ng/mL.

In another aspect, the invention pertains to a BMP6 anagonist for use intreating a patient having anemia, characterized in that atherapeutically effective amount of the BMP6 antagonist is to beadministered to the patient on the basis of a biological sample from thepatient having a ferritin level of ≥500 ng/mL.

In another aspect, the invention pertains to a BMP6 antagonist for usein treating a patient having anemia, characterized in that:

-   a) the patient is to be selected for treatment with the BMP6    antagonist on the basis of a biological sample from the patient    having a ferritin level of ≤2000 ng/mL; and-   b) thereafter, a therapeutically effective amount of the BMP6    antagonist s to be administered to the patient. In embodiments, the    ferritin level is ≥500 ng/mL.

In another aspect, the invention pertains to a BMP6 antagonist for usein treating a patient having anemia, characterized in that:

-   a) the patient is to be selected for treatment with the BMP6    antagonist on the basis of a biological sample from the patient    having a ferritin level of ≥500 ng/mL; and-   b) thereafter, a therapeutically effective amount of the BMP6    antagonist is to be administered to the patient.

In another aspect, the invention pertains to a BMP6 antagonist for usein treating a patient having anemia, characterized in that:

-   a) a biological sample from the patient is to be assayed for    ferritin; and-   b) a therapeutically effective amount of the BMP6 antagonist is to    be selectively administered to the patient on the basis of the    biological sample from the patient having a ferritin level of ≤2000    ng/mL. In embodiments, the ferritin level is ≥500 ng/mL.

In another aspect, the invention pertains to a BMP6 antagonist for usein treating a patient having anemia, characterized in that:

-   a) a biological sample from the patient is to be assayed for    ferritin; and-   b) a therapeutically effective amount of the BMP6 antagonist is to    be selectively administered to the patient on the basis of the    biological sample from the patient having a ferritin level of ≤500    ng/mL.

In another aspect, the invention pertains to a BMP6 antagonist for usein treating a patient having anemia, characterized in that:

-   a) a biological sample from the patient is to be assayed for    ferritin;-   b) the patient is selected for treatment with the BMP6 antagonist on    the basis of the biological sample from the patient having a    ferritin level of 2000 ng/mL; and-   c) a therapeutically effective amount of the BMP6 antagonist is to    be selectively administered to the patient. In embodiments, the    ferritin level is ≥500 ng/mL.

In another aspect, the invention pertains to a BMP6 antagonist for usein treating a patient having anemia, characterized in that:

-   a) a biological sample from the patient is to be assayed for    ferritin;-   b) the patient is selected for treatment with the BMP6 antagonist on    the basis of the biological sample from the patient having a    ferritin level of ≥500 ng/mL; and-   c) a therapeutically effective amount of the BMP6 antagonist is to    be selectively administered to the patient.

In another aspect, the invention pertains to method of predicting thelikelihood that a patient having anemia will respond to treatment with aBMP6 antagonist, including assaying a biological sample from the patientfor ferritin, wherein a ferritin level of 2000 ng/mL is indicative of anincreased likelihood the patient will respond to treatment with the BMP6antagonist. In embodiments, the ferritin level is ≥500 ng/mL.

In another aspect, the invention pertains to method of predicting thelikelihood that a patient having anemia will respond to treatment with aBMP6 antagonist, including assaying a biological sample from the patientfor ferritin, wherein a ferritin level of ≥500 ng/mL is indicative of anincreased likelihood the patient will respond to treatment with the BMP6antagonist.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the method or use further includes the step ofobtaining the biological sample from the patient, wherein the step ofobtaining is performed prior to the step of assaying.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the ferritin level is ferritin protein level.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the step of assaying includes a techniqueselected from the group consisting of an immunoassay,immunohistochemistry, ELISA, flow cytometry, Western blot, HPLC, andmass spectrometry.

In another aspect, the invention provides a method for producing atransmittable form of information for predicting the responsiveness of apatient having anemia to treatment with a BMP6 antagonist, including:

-   a) determining an increased likelihood of the patient responding to    treatment with the BMP6 antagonist based on the presence of a    ferritin level of ≤2000 ng/mL in a biological sample from the    patient; and-   b) recording the result of the determining step on a tangible or    intangible media form for use in transmission. In embodiments, the    ferritin level is ≥500 ng/mL.

In another aspect, the invention provides a method for producing atransmittable form of information for predicting the responsiveness of apatient having anemia to treatment with a BMP6 antagonist, including:

-   a) determining an increased likelihood of the patient responding to    treatment with the BMP6 antagonist based on the presence of a    ferritin level of ≥500 ng/mL in a biological sample from the    patient; and-   b) recording the result of the determining step on a tangible or    intangible media form for use in transmission.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the patient has anemia. In embodiments, theanemia is anemia associated with chronic disease. In embodiments, thechronic disease is chronic kidney disease, cancer or inflammation.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the patient is being or has been treated withan erythropoiesis stimulating agent (ESA), for example, erythropoietin(EPO).

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anemia is EPO-hyporesponsive anemia.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anemia is iron-restricted anemia, forexample, functional iron-restricted anemia.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the patient is a chronic hemodialysis patient.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the method or use further includes reducing thepatient's iron dose requirement, reducing the patient's EPO doserequirement, or reducing both the patient's iron dose requirement andthe patient's EPO dose requirement, relative to said EPO doserequirement and/or iron dose requirement in the absence of treatmentwith the therapeutically effective amount of the BMP6 antagonist. Inembodiments, including in embodiments of any of the aforementionedaspects and embodiments, the method or use further includes or resultsin a reduction in the patient's ESA resistance index (ERI).

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the biological sample is synovial fluid, blood,serum, feces, plasma, urine, tear, saliva, cerebrospinal fluid, aleukocyte sample or a tissue sample.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the biological sample is serum or blood.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the biological sample is serum.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the BMP6 antagonist is a BMP6 binding molecule.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the BMP6 antagonist is an anti-BNIP6 antibodyor antigen-binding fragment thereof for example, as described in Table 1or Table 14.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes:

-   (a) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 69, 70 and 71,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:    79, 80 and 81, respectively;-   (b) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 72, 73 and 74,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:    82, 83 and 84, respectively;-   (c) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 29, 30 and 31,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ NOs: 39,    40 and 41, respectively;-   (d) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:    42, 43 and 44, respectively;-   (e) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 49, 50 and 51,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:    59, 60 and 61, respectively;-   (f) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 52, 53 and 54,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:    62, 63 and 64, respectively;-   (g) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 9, 10 and 11,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:    19, 20 and 21, respectively; or-   (h) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 12, 13 and 14,    respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:    22, 23 and 24, respectively.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes:

-   (a) A VH sequence of SEQ ID NO: 75;-   (b) A VH sequence of SEQ ID NO: 35;-   (c) A VH sequence of SEQ ID NO: 55; or-   (d) A VH sequence of SEQ ID NO: 15.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes:

-   (a) A VL sequence of SEQ ID NO: 85;-   (b) A VL sequence of SEQ ID NO: 45;-   (c) A VL sequence of SEQ ID NO: 65; or-   (d) A VL sequence of SEQ ID NO: 25.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes:

-   (a) A VH sequence of SEQ ID NO: 75; and a VL sequence of SEQ ID NO:    85;-   (b) A VH sequence of SEQ ID NO: 35; and a VL sequence of SEQ ID NO:    45;-   (c) A VH sequence of SEQ ID NO: 55; and a VL sequence of SEQ ID NO:    65; or-   (d) A VH sequence of SEQ ID NO: 15; and a VL sequence of SEQ ID NO:    25.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes:

-   (a) A heavy chain sequence of SEQ ID NO: 77;-   (b) A heavy chain sequence of SEQ ID NO: 37;-   (c) A heavy chain sequence of SEQ ID NO: 57; or-   (d) A heavy chain sequence of SEQ NO: 17.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes:

-   (a) A light chain sequence of SEQ ID NO: 87;-   (b) A light chain sequence of SEQ ID NO: 47;-   (c) A light chain sequence of SEQ ID NO: 67; or-   (d) A light chain sequence of SEQ II) NO: 27.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes:

-   (a) A heavy chain sequence of SEQ ID NO: 77; and a light chain    sequence of SEQ ID NO: 87;-   (b) A heavy chain sequence of SEQ ID NO: 37; and a light chain    sequence of SEQ ID NO: 47;-   (c) A heavy chain sequence of SEQ ID NO: 57; and a light chain    sequence of SEQ ID NO: 67; or-   (d) A heavy chain sequence of SEQ ID NO: 17; and a light chain    sequence of SEQ ID NO: 27.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof binds human BMP6 with a KD of ≤1 nM.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof binds human BMP6 with a KD of ≤0.1 nM.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof has at least about 100-fold greater affinity for humanBMP6 than human BMP7.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof has at least about 100-fold greater affinity for humanBMP6 than human BMP2, human BMPS, or human BMP7.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof has at least about 500-fold greater affinity for humanBMP6 than human BMP2, human BMP5, or human BMP7.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof has no detectable binding to human BMP2 and/or BMP7 inan ELISA.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof includes a scaffold selected from an IgM and an IgG.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof is an IgG selected from an IgG1, an IgG2, and IgG3 oran IgG4.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof is selected from the group consisting of: a monoclonalantibody, a chimeric antibody, a single chain antibody, a Fab and ascFv.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof is a component of an immunoconjugate.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof has altered effector function through mutation of theFc region.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the anti-BMP6 antibody or antigen-bindingfragment thereof binds to a human BMP6 epitope including, e.g.,consisting of, the sequence

(SEQ ID NO: 98) QTLVHLMNPEYVPKP.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the antibody or antigen-binding fragmentthereof is administered at a dose ranging from 0.001 mg/kg to 0.1 mg/kg.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the antibody or antigen-binding fragmentthereof is administered at a dose ranging from 0.0063 to 0.1 mg/kg.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the antibody or antigen-binding fragmentthereof is administered at a dose of 0.001 mg/kg, 0.0016 mg/kg, 0.0025mg/kg, 0.0040 mg/kg, 0.0063 mg/kg, 0.01 mg/kg, 0.016 mg/kg, 0.025 mg/kg,0.040 mg/kg, 0.063 mg/kg, or 0.1 mg/kg.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the antibody or antigen-binding fragmentthereof is administered intravenously or subcutaneously.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the antibody or antigen-binding fragmentthereof is administered intravenously.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the administration is by infusion over a periodof about 30 to about 60 minutes.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the ferritin level is ≤1900 ng/mL, ≤1800 ng/mL,≤1700 ng/mL, ≤1600 ng/mL, ≤1500 ng/mL, ≤1400 ng/mL, ≤1300 ng/mL, ≤1200ng/mL, ≤1100 ng/mL, ≤1000 ng/mL, ≤900 ng/mL, ≤800 ng/mL, ≤700 ng/mL,≤600 ng/mL, or ≤500 ng/mL.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the ferritin level is ≤1500 ng/mL. Inembodiments, including in embodiments of any of the aforementionedaspects and embodiments, the ferritin level is ≤1000 ng/mL.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the ferritin level is ≥about 200 ng/mL, ≥about250 ng/mL, ≥about 300 ng/mL, ≥about 350 ng/mL, ≥about 400 ng/mL, ≥about450 ng/mL, ≥about 500 ng/mL, ≥about 600 ng/mL, ≥about 700 ng/mL, ≥about800 ng/mL, ≥about 900 ng/mL, ≥about 1000 ng/mL, ≥about 1100 ng/mL,≥about 1200 ng/mL, ≥about 1300 ng/mL, ≥about 1400 ng/mL, ≥about 1500ng/mL, ≥about 1600 ng/mL, ≥about 1700 ng/mL, ≥about 1800 ng/mL, or≥about 1900 ng/mL.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the ferritin level is ≥about 500 ng/mL, ≥about600 ng/mL, ≥about 700 ng/mL, ≥about 800 ng/mL, ≥about 900 ng/mL, ≥about1000 ng/mL, ≥about 1100 ng/mL, ≥about 1200 ng/mL, ≥about 1300 ng/mL, or≥about 1400 ng/mL.

In embodiments, including in embodiments of any of the aforementionedaspects and embodiments, the ferritin level is ≥about 500 ng/mL.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains.

“BMP6”, as used herein, means the protein Bone Morphogenetic Protein 6(BMP6) or a gene or nucleic acid encoding BMP6. Hahn et al. 1992Genomics 14: 759-62; Sauermann et al. 1993 J. Neurosci. Res. 33: 142-7;NCBI Gene ID: 654. BMP6 is also known as: BMP-6; VGR; VGR1; ExternalIDs: OMIM: 112266 MGI: 88182; HomoloGene: 1300; GeneCards: BMP6 Gene.Orthologs: Species: Human: Entrez: 654; Ensembl: ENSG00000153162;UniProt: P22004; RefSeq (mRNA): NM_001718; RefSeq (protein): NP_001709;Location (UCSC): Chr 6: 7.73-7.88 Mb; Species: Mouse: Entrez: 12161;Ensembl: ENSMUSG00000039004; UniProt: P20722; RefSeq (mRNA): NM_907556;RefSeq (protein): NP_031582; Location (UCSC): Chr 13: 38.35-38.5 Mb. Asdescribed herein, an antibody antigen-binding fragment thereof whichhinds to BMP6 binds to BMP6 protein.

“BMP2”, as used herein, means the protein Bone Morphogenetic Protein 2(BMP2) or a gene or nucleic acid encoding BMP2. BMP2 is also known as:BDA2; and BMP2A; External IDs OMIM: 112261 MGI: 88177 HomoloGene: 926GeneCards: BMP2 Gene. Species: Human; Entrez: 650; Ensembl:ENSG00000125845; UniProt: P12643; RefSeq (mRNA): NM_001200; RetSeq(protein): NP_01191; Location (UCSC): Chr 20: 6.75-6.76 Mb. Species:Mouse; Entrez: 12156; Ensembl: ENSMUSG00000027358; UniProt: P21274;RefSeq (mRNA): NM_007553; RefSeq (protein): NP_031579; Location (UCSC):Chr 2: 133.55-133.56 Mb. As described herein, an antibodyantigen-binding fragment thereof which binds to BMP2 binds to BMP2protein.

“BMP5”, as used herein, means the protein Bone Morphogenetic Protein 5(BMP5) or a gene or nucleic acid encoding BMP5. BMP5 is also known as:MGC34244; External IDs OMIM: 112265 MGI: 88181 HomoloGene: 22412GeneCards: BMP5 Gene. Species: Human; Entrez: 653; Ensembl:ENSG00000112175; UniProt: P22003; RefSeq (mRNA): NM_021073; RefSeq(protein): NP_066551; Location (UCSC): Chr 6: 55.62-55.74 Mb. Species:Mouse; Entrez: 12160; Ensembl: ENSMUSG00000032179; UniProt: P49003;RefSeq (mRNA): NM_007555; RefSeq (protein): NP_031581; Location (UCSC):Chr 9: 75.78-75.9 Mb. As described herein, an antibody antigen-bindingfragment thereof which binds to BMPS binds to BMPS protein.

“BMP7”, as used herein, means the protein Bone Morphogenetic Protein 7(BMP7) or a gene or nucleic acid encoding BMP7. BMP7 is also known as:osteogenic protein-1; OP-1; External IDs OMIM: 112267 MGI: 103302HomoloGene: 20410 GeneCards: BMP7 Gene. Species: Human; Entrez: 655;Ensembl: ENSG00000101144; UniProt: P18075; RefSeq (mRNA): NM_001719;RefSeq (protein): NP_001710; Location (UCSC): Chr 20: 55.74-55.84 Mb.Species: Mouse; Entrez: 12162; Ensembl: ENSMUSG00000008999; UniProt:P23359; RefSeq (mRNA): NM_007557; RefSeq (protein): NP_031583; Location(UCSC): Chr 2: 172.87-172.94 Mb. As described herein, an antibodyantigen-binding fragment thereof which binds to BMP7 binds to BMP7protein.

“Hepcidin” means the gene Hepcidin or the protein Hepcidin, a peptidehormone. Hepcidin is also known as: HAMP (Hepcidin anti-microbialprotein or peptide); HEPC; HFE2B; LEAP1 (LEAP-1); PLTR; OMIM: 606464;HomoloGene: 81623; GeneCards: HAMP Gene; Entrez 57817; EnsemblENSG00000105697; UniProt P81172; RefSeq (mRNA) NM_021175; RefSeq(protein) NP_066998; Location (UCSC) Chr 19: 35.77-35.78 Mb. Krause etal. FEBS Lett. 480: 147-150; and Pigeon et al. 2001 J. Biol. Chem. 276:7811-9. See also: Ganz 2003 Blood 102: 783-8; Roy et al. 2005 Curr.Opin. Hemat. 12: 107-111; Fleming et al. 2006 Semin. Liver Dix. 25:411-9; Park et al. 2001 J. Biol. Chem. 276: 7806-10; Majore et al. 2002Haematologica 87: 221-2; Kluver et al. 2002 J. Pept. Res. 59: 241-8;Hunter et al. 2002 J. Biol. Chem. 277: 37597-603; Weinstein et al. 2003Blood 100: 3776-81; Nemeth et al. 2003 Blood 101: 2461-3; Roetto et al.2003 Nat. Genet. 33: 21-2; Strausberg et al. 2003 Proc. Natl. Acad. SciUSA 99: 16899-903; Gehrke et al. 2003 Blood 102: 371-6;Merryweather-Clarke et al. 2004 Human Mol. Genet, 12:2241-7; Clark etal. 2003 Genome Res. 13: 2265-70; Roetto et al. 2004 Blood 103: 2407-9;Jacolot et al. 2004 Blood 103: 2835-40; and Ota et al. 2004 Nat. Genet.36: 40-45.

“BMP6 antagonist,” as used herein refers to a molecule capable ofantagonizing (e.g., reducing, inhibiting, decreasing, delaying) BMP6function, expression and/or signalling (e.g., by blocking the binding ofBMP6 to a BMP6 receptor). Non-limiting examples of BMP6 antagonistsinclude BMP6 binding molecules and BMP6 receptor binding molecules. Insome embodiments of the disclosed methods, regimens, kits, processes,uses and compositions, a BMP6 antagonist is employed.

“BMP6 binding molecule,” as used herein, refers to any molecule capableof binding to the human BMP6 antigen either alone or associated withother molecules. The binding reaction may be shown by standard methods(qualitative assays) including, for example, a binding assay,competition assay or a bioassay for determining the inhibition of BMP6binding to a BMP6 receptor or any kind of binding assays, with referenceto a negative control test in which an antibody of unrelatedspecificity, but ideally of the same isotype is used. Non-limitingexamples of BMP6 binding molecules include small molecules, BMP6receptor decoys, and antibodies that bind to BMP6 as produced by B cellsor hybridomas and chimeric, CDR-grafted or human antibodies or anyfragment thereof, e.g., F(ab′)2 and Fab fragments, as well as singlechain or single domain antibodies. Preferably the BMP6 binding moleculeantagonizes (e.g., reduces, inhibits, decreases, delays) BMP6 function,expression and/or signalling. In some embodiments of the disclosedmethods, regimens, kits, processes, uses and compositions, an BMP6binding molecule is employed.

By “BMP6 receptor binding molecule” is meant any molecule capable ofbinding to a human BMP6 receptor either alone or associated with othermolecules. The binding reaction may be shown by standard methods(qualitative assays) including, for example, a binding assay,competition assay or a bioassay for determining the inhibition of BMP6receptor binding to BMP6 or any kind of binding assays, with referenceto a negative control test in which an antibody of unrelatedspecificity, but ideally of the same isotype is used. Non-limitingexamples of BMP6 receptor binding molecules include small molecules.BMP6 decoys, and antibodies to the BMP6 receptor as produced by B cellsor hybridomas and chimeric, CDR-grafted or human antibodies or anyfragment thereof, e.g., F(ab′)2 and Fab fragments, as well as singlechain or single domain antibodies. Preferably the BMP6 receptor bindingmolecule antagonizes (e.g., reduces, inhibits, decreases, delays) BMP6function, expression and/or signaling. In some embodiments of thedisclosed methods, regimens, kits, processes, uses and compositions, anBMP6 receptor binding molecule is employed.

“Anemia”, as used herein, means a decrease in the number of red bloodcells, or a decrease in the amount of hemoglobin or iron in the blood,with a decreased ability of the blood to carry oxygen.

“EPO resistance index” or “ERI”, as used herein interchangeably, meansthe change in ESA, e.g., EPO, dose (Units per kg body weight) as afunction of hemoglobin level. In embodiments, the ESA dose/hemoglobinlevel is measured weekly. In embodiments, the ESA dose/hemoglobin levelis measured monthly. In embodiments, the ESA dose/hemoglobin level iscompared over multiple measurement to arrive at the ERI.

Anemia can be diagnosed using any method known in the art, including, asa non-limiting example, in men based on a hemoglobin of less than about130 to 140 g/L (13 to 14 g/dL) and in women, less than about 120 to 130(12 to 13 g/dL). Janz et al. 2013 Emerg. Med. Pract. 15: 1-15; and Smith2010 Am. J. Man. Care 16 Supp. S59-66.

As used herein, the terms “BMP6 antibody,” “anti-human BMP6 antibody,”“BMP6-binding antibody”, “BMP6 antagonist antibody” and the like (andantigen-binding fragments thereof) include antibodies (andantigen-binding fragments thereof) which bind to the protein BMP6.

The terms “antibody”, “antigen-binding fragment thereof”, “antigenbinding portion,” and the like, as used herein, include whole antibodiesand any antigen-binding fragment (i.e., “antigen-binding portion”) orsingle chains thereof. A naturally occurring “antibody” is aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain is comprisedof a heavy chain variable region (abbreviated herein as VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarily determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRsarranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g, effector cells) and the first component(C1q) of the classical complement system.

The terms “antigen-binding fragment”, “antigen-binding fragmentthereof,” “antigen binding portion” of an antibody, and the like, asused herein, refer to one or more fragments of an intact antibody thatretain the ability to specifically bind to a given antigen (e.g., BMP6).Antigen binding functions of an antibody can be performed by fragmentsof an intact antibody. Examples of binding fragments encompassed withinthe term “antigen binding portion” of an antibody include a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; a F(ab)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; an Fdfragment consisting of the VH and CH1 domains; an Fv fragment consistingof the VL and VH domains of a single arm of an antibody; a single domainantibody (dAb) fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a VH domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by an artificial peptide linker that enables them to be made asa single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet at., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl.Acad. Sci. 85:5879-5883). Such single chain antibodies include one ormore “antigen binding portions” of an antibody. These antibody fragmentsare obtained using conventional techniques known to those of skill inthe art, and the fragments are screened for utility in the same manneras are intact antibodies.

Antigen binding portions can also be incorporated into single domainantibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005,Nature Biotechnology, 23, 9, 1126-1136). Antigen binding portions ofantibodies can be grafted into scaffolds based on polypeptides such asFibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describesfibronectin polypeptide monobodies).

Antigen binding portions can be incorporated into single chain moleculescomprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et at., 1995 Protein Eng. 8 (10):1057-1062; andU.S. Pat. No. 5,641,870).

As used herein, the term “Affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity.

As used herein, the term “Avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalency of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an alpha carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid,

The term “binding specificity” as used herein refers to the ability ofan individual antibody combining site to react with only one antigenicdeterminant. The combining site of the antibody is located in the Fabportion of the molecule and is constructed from the hypervariableregions of the heavy and light chains. Binding affinity of an antibodyis the strength of the reaction between a single antigenic determinantand a single combining site on the antibody. It is the sum of theattractive and repulsive forces operating between the antigenicdeterminant and the combining site of the antibody.

Specific binding between two entities means a binding with anequilibrium constant (KA or K_(A)) of at least 1×10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹M⁻¹, 10¹⁰ M⁻¹, or 10¹¹ M⁻¹. The phrase “specifically (or selectively)binds” to an antibody (e.g., BMP6-binding antibody) refers to a bindingreaction that is determinative of the presence of a cognate antigen(e.g., a human BMP6 protein) in a heterogeneous population of proteinsand other biologics. In addition to the equilibrium constant (KA) notedabove, an BMP6-binding antibody of the invention typically also has adissociation rate constant (Kd or KD or K_(D)) of about 1×10⁻² s⁻¹,1×10⁻³ s⁻¹, or lower, and binds to BMP6 with an affinity that is atleast two-fold greater than its affinity for binding to a non-specificantigen (e.g., BMP2, BMP5 or BMP7). The phrases “an antibody recognizingan antigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

Specific binding between two entities means a binding with anequilibrium constant (KA) (kon/koff) of at least 10²M⁻¹, at least5×10²M⁻¹, at least 10³M⁻¹, at least 5×10³M⁻¹, at least 10⁴M⁻¹ at least5×10⁴M⁻¹, at least 10⁵M⁻¹, at least 5×10⁵M⁻¹, at least 10⁶M⁻¹, at least5×10⁶M⁻¹, at least 10⁷M⁻¹, at least 5×10⁷M⁻¹, at least 10³M⁻¹, at least5×10⁸M⁻¹, at least 10⁹M⁻¹, at least 5×10⁹M⁻¹, at least 10¹⁰M⁻¹, at least5×10¹⁰M⁻¹, at least 10¹¹M⁻¹, at least 5×10¹¹M⁻¹, at least 10¹²M⁻¹, atleast 5×10¹²M⁻¹, at least 10¹³M⁻¹, at least 5×10¹³M⁻¹, at least 10¹⁴M⁻¹,at least 5×10¹⁴M⁻¹, at least 10¹⁵M⁻¹, or at least 5×10¹⁵M⁻¹.

The term “chimeric antibody” (or antigen-binding fragment thereof) is anantibody molecule (or antigen-binding fragment thereof) in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, es., an enzyme, toxin, hormone,growth factor, drug, etc.; or (h) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity. For example, a mouseantibody can be modified by replacing its constant region with theconstant region from a human immunoglobulin. Due to the replacement witha human constant region, the chimeric antibody can retain itsspecificity in recognizing the antigen while having reduced antigenicityin human as compared to the original mouse antibody.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, Creighton, Proteins (1984)). In one embodiment, theterm “conservative sequence modifications” are used to refer to aminoacid modifications that do not significantly affect or alter the bindingcharacteristics of the antibody containing the amino acid sequence.

The term “blocks” as used herein refers to stopping or preventing aninteraction or a process, e.g., stopping ligand-dependent orligand-independent signaling.

The term “recognize” as used herein refers to an antibodyantigen-binding fragment thereof that finds and interacts (e.g., binds)with its conformational epitope. The terms “cross-block”,“cross-blocked”, “cross-blocking”, “compete”, “cross compete” andrelated terms are used interchangeably herein to mean the ability of anantibody or other binding agent to interfere with the binding of otherantibodies or binding agents to BMP6 in a standard competitive bindingassay.

The ability or extent to which an antibody or other binding agent isable to interfere with the binding of another antibody or bindingmolecule to BMP6, and therefore whether it can be said to cross-blockaccording to the invention, can be determined using standard competitionbinding assays. One suitable involves the use of the Biacore technology(e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)),which can measure the extent of interactions using surface plasmonresonance technology. Another assay for measuring cross-blocking uses anELISA-based approach.

The term “neutralizes” means that an antibody, upon binding to itstarget, reduces the activity, level or stability of the target; e.g., aBMP6 antibody, upon binding to BMP6 neutralizes BMP6 by at leastpartially reducing an activity, level or stability of BMP6, such assignaling or its role in hepcidin levels and anemia.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or otherwise interacting with a molecule.Epitopic determinants generally consist of chemically active surfacegroupings of molecules such as amino acids BMP6or carbohydrate or sugarside chains and can have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics. An epitopemay be “linear” or “conformational.”

The term “linear epitope” refers to an epitope with all of the points ofinteraction between the protein and the interacting molecule (such as anantibody) occur linearally along the primary amino acid sequence of theprotein (continuous). As used herein, the term “high affinity” for anIgG antibody refers to an antibody having a KD of 10⁻⁸ M or less, 10⁻⁹ Mor less, or 10⁻¹⁰ M, or 10⁻¹¹ M or less for a target antigen, e.g.,BMP6. However, “high affinity” binding can vary for other antibodyisotypes. For example, “high affinity” binding for an IgM isotype refersto an antibody having a KD of 10⁻⁷ M or less, or 10⁻⁸ M or less.

The term “human antibody” (or antigen-binding fragment thereof), as usedherein, is intended to include antibodies (and antigen-binding fragmentsthereof) having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences. The human antibodies andantigen-binding fragments thereof of the invention may include aminoacid residues not encoded by human sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo).

The phrases “monoclonal antibody” or “monoclonal antibody composition”(or antigen-binding fragment thereof) as used herein refers topolypeptides, including antibodies, antibody fragments, bispecificantibodies, etc. that have substantially identical to amino acidsequence or are derived from the same genetic source. This term alsoincludes preparations of antibody molecules of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope.

The term “human monoclonal antibody” (or antigen-binding fragmentthereof) refers to antibodies (and antigen-binding fragments thereof)displaying a single binding specificity which have variable regions inwhich both the framework and CDR regions are derived from humansequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The phrase “recombinant human antibody” (or antigen-binding fragmentthereof), as used herein, includes all human antibodies (andantigen-binding fragments thereof) that are prepared, expressed, createdor isolated by recombinant means, such as antibodies isolated from ananimal a mouse) that is transgenic or transchromosomal for humanimmunoglobulin genes or a hybridoma prepared therefrom, antibodiesisolated from a host cell transformed to express the human antibody,e.g., from a transfectoma, antibodies isolated from a recombinant,combinatorial human antibody library, and antibodies prepared,expressed, created or isolated by any other means that involve splicingof all or a portion of a human immunoglobulin gene, sequences to otherDNA sequences. Such recombinant human antibodies have variable regionsin which the framework and CDR regions are derived from human germlineimmunoglobulin sequences. In one embodiment, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

A “humanized” antibody (or antigen-binding fragment thereof), as usedherein, is an antibody (or antigen-binding fragment thereof) thatretains the reactivity of a non-human antibody while being lessimmunogenic in humans. This can be achieved, for instance, by retainingthe non-human CDR regions and replacing the remaining parts of theantibody with their human counterparts (i.e., the constant region aswell as the framework portions of the variable region). See, e.g.,Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984;Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al.,Science, 239:1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991;and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of humanengineering technology include, but is not limited to Xoma technologydisclosed in U.S. Pat. No. 5,766,886.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length. Optionally, the identityexists over a region that is at least 50 nucleotides (or 10 amino acids)in length, or more preferably over a region that is 100 to 500 or 1000or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are desimated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Brent etal., Current Protocols in Molecular Biology, John Wiley & Sons. Inc.(ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefrills off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (N) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17, 1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol. Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5. or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “isolated antibody” (or antigen-binding fragment thereof), asused herein, refers to an antibody (or antigen-binding fragment thereof)that is substantially free of other antibodies having differentantigenic specificities (e.g, an isolated antibody that specificallybinds BMP6 is substantially free of antibodies that specifically bindantigens other than BMP6). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “isotype” refers to the antibody class (e.g., IgM, IgE, IgGsuch as IgG1 or IgG4) that is provided by the heavy chain constantregion genes. Isotype also includes modified versions of one of theseclasses, where modifications have been made to after the Fc function,for example, to enhance or reduce effector functions or binding to Fcreceptors.

The term “Kassoc” or “Ka” or “KA” or “K_(A)”, as used. herein, isintended to refer to the association rate of a particularantibody-antigen interaction, whereas the term “Kdis” or “Kd,” as usedherein, is intended to refer to the dissociation rate of a particularantibody-antigen interaction. In one embodiment, the term “K_(D)”, asused herein, is intended to refer to the dissociation constant, which isobtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as amolar concentration (M). K_(D) values for antibodies can be determinedusing methods well established in the art. A method for determining theK_(D) of an antibody is by using surface plasmon resonance, or using abiosensor system such as a Biacore® system.

The terms “monoclonal antibody” (or antigen-binding fragment thereof) or“monoclonal antibody (or antigen-binding fragment thereof) composition”as used herein refer to a preparation of an antibody molecule (orantigen-binding fragment thereof) of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem.260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98,1994).

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, it refers tothe functional relationship of a transcriptional regulatory sequence toa transcribed sequence. For example, a promoter or enhancer sequence isoperably linked to a coding sequence if it stimulates or modulates thetranscription of the coding sequence in an appropriate host cell orother expression system. Generally, promoter transcriptional regulatorysequences that are operably linked to a transcribed sequence arcphysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory sequences, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO)or a human cell. The optimized nucleotide sequence is engineered toretain completely or as much as possible the amino acid sequenceoriginally encoded by the starting nucleotide sequence, which is alsoknown as the “parental” sequence. The optimized sequences herein havebeen engineered to have codons that are preferred in mammalian cells.However, optimized expression of these sequences in other eukaryoticcells or prokaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “recombinant human antibody” (or antigen-binding fragmentthereof), as used herein, includes all human antibodies (andantigen-binding fragments thereof) that are prepared, expressed, createdor isolated by recombinant means, such as antibodies isolated from ananimal (e.g., a mouse) that is transgenic or transchromosomal for humanimmunoglobulin genes or a hybridoma prepared therefrom, antibodiesisolated from a host cell transformed to express the human antibody,e.g., from a transfectoma, antibodies isolated from a recombinant,combinatorial human antibody library, and antibodies prepared,expressed, created or isolated by any other means that involve splicingof all or a portion of a human immunoglobulin gene, sequences to otherDNA sequences. Such recombinant human antibodies have variable regionsin which the framework and CDR regions are derived from human germlineimmunoglobulin sequences. In one embodiment, however, such recombinanthuman antibodies can be subjected to in vitro mutagenesis (or, when ananimal transgenic for human Ig sequences is used, in vivo somaticmutagenesis) and thus the amino acid sequences of the VH and VL regionsof the recombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “recombinant host cell” (or simply “host cell”) refers to acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, and reptiles.Except when noted, the terms “patient” or “subject” are used hereininterchangeably.

The term “treating” includes the administration of compositions orantibodies to prevent or delay the onset of the symptoms, complications,or biochemical indicia of a disease (e.g., anemia), alleviating thesymptoms or arresting or inhibiting further development of the disease,condition, or disorder. Treatment may be prophylactic (to prevent ordelay the onset of the disease, or to prevent the manifestation ofclinical or subclinical symptoms thereof) or therapeutic suppression oralleviation of symptoms after the manifestation of the disease.

The term “vector” is intended to refer to a polynucleotide moleculecapable of transporting another polynucleotide to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “hematocrit” or “haematocrit”, as used herein, is also known asa packed cell volume (PCV) or erythrocyte volume fraction (EVF) and isthe volume (%) of red blood cells in blood. This is normally about 45%for men and about 50% for women. It is considered an integral part of aperson's complete blood count results, along with hemoglobinconcentration, white blood count, and platelet count. In one embodiment,anemia refers to an abnormally low hematocrit, as opposed topolycythemia, which is an abnormally high hematocrit.

The term “assaying” is used to refer to the act of identifying,screening, probing, testing measuring or determining, which act may beperformed by any conventional means. For example, a sample may beassayed for the presence of a particular genetic or protein marker byusing an ELISA assay, a Northern blot, imaging, serotyping, cellulartyping, gene sequencing, phenotyping, haplotyping, immunohistochemistry,western blot, mass spectrometry, etc. The term “detecting” (and thelike) means the act of extracting particular information from a givensource, which may be direct or indirect. In some embodiments of thepredictive methods disclosed herein, the presence or level of a giventhing (e.g., level of protein, etc.) is detected in a biological sampleindirectly, e.g., by querying a database. The terms “assaying” and“determining” contemplate a transformation of matter, e.g., atransformation of a biological sample, e.g., a blood sample or othertissue sample, from one state to another by means of subjecting thatsample to physical testing.

The term “obtaining” means to procure, e.g., to acquire possession of inany way, e.g., by physical intervention (e.g., biopsy, blood draw) ornon-physical intervention (e.g., transmittal of information via aserver), etc.

The phrase “assaying a biological sample . . . ” and the like, is usedto mean that a sample may be tested (either directly or indirectly) foreither the presence or level of a given marker (e.g., protein). It willbe understood that, in a situation where the level of a substancedenotes a probability, then the level of such substance may be used toguide a therapeutic decision. For example, one may determine the levelof ferritin in a patient by assaying for its presence in by quantitativeor relatively-quantitative means (e.g., levels relative to the levels inother samples). The disclosed methods involve, inter alia, determiningthe level of a particular marker, e.g., ferritin, in a patient.

As used herein, “selecting” and “selected” in reference to a patient isused to mean that a particular patient is specifically chosen from alarger group of patients on the basis of (due to) the particular patienthaving a predetermined criteria, e.g., the patient has a particularlevel of ferritin, e.g., as described herein. Similarly, “selectivelytreating” refers to providing treatment to a patient having a particulardisease, where that patient is specifically chosen from a larger groupof patients on the basis of the particular patient having apredetermined criteria, e.g., an anemia patient specifically chosen fortreatment due to the patient having a particular ferritin level, e.g.,as described herein. Similarly, “selectively administering” refers toadministering a drug to a patient that is specifically chosen from alarger group of patients on the basis of (due to) the particular patienthaving a predetermined criteria, e.g., a particular genetic or otherbiological marker, e.g., a particular ferritin level, e.g., as describedherein. By selecting, selectively treating and selectivelyadministering, it is meant that a patient is delivered a personalizedtherapy based on the patient's particular biology, rather than beingdelivered a standard treatment regimen based solely on the patienthaving a particular disease. Selecting, in reference to a method oftreatment as used herein, does not refer to fortuitous treatment of apatient that has a particular marker, but rather refers to thedeliberate choice to administer a BMP6 antagonist to a patient based onthe patient having a particular marker, e.g., a particular ferritinlevel, e.g., as described herein. Thus, selective treatment differs fromstandard treatment, which delivers a particular drug to all patients,regardless of their marker status.

As used herein, “predicting” indicates that the methods described hereinprovide information to enable a health care provider to determine thelikelihood that an individual having a disease, e.g., anemia, willrespond to or will respond more favorably to treatment with BMP6 bindingmolecule. It does not refer to the ability to predict response with 100%accuracy. Instead, the skilled artisan will understand that it refers toan increased probability.

As used herein. “likelihood” and “likely” is a measurement of howprobable an event is to occur. It may be used interchangably with“probability”. Likelihood refers to a probability that is more thanspeculation, but less than certainty. Thus, an event is likely if areasonable person using common sense, training or experience concludesthat, given the circumstances, an event is probable. In someembodiments, once likelihood has been ascertained, the patient may betreated (or treatment continued, or treatment proceed with a dosageincrease with the BMP6 binding molecule or the patient may not betreated (or treatment discontinued, or treatment proceed with a lowereddose) with the BMP6 binding molecule.

The phrase “increased likelihood” refers to an increase in theprobability that an event will occur. For example, some methods hereinallow prediction of whether a patient will display an increasedlikelihood of responding to treatment with a BMP6 binding molecule or anincreased likelihood of responding better to treatment with a BMP6binding molecule in comparison to a patient having the same or similardisease who does not have the marker (e.g., the level of ferritin asdescribed herein).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows inhibition of BMP activity by antagonist antibodies 5, 6and 7 in reporter gene assay. Activity against BMP2, BLIPS, BMP6, andBMP7 is shown.

FIG. 1B shows an ELISA binding assay testing Antibody 7 binding to humanBMP6, human BMP7, human BMP5, mouse BMP6, hBaffR, BSA and Neu. In thisfigure and various other figures, and elsewhere in the Specification, Ab5=Antibody 5; Ab 6=Antibody 6; and Ab 7=Antibody 7.

FIG. 2 shows the pharmacodynamics profiles of single dose rat triage PKstudy. Antibodies 5, 6 and 7 were used. Serum hepcidin and iron levelswere measured at 1 hr, 6 hr, 1, 2, 4, 8, 16 days post dose (10 mg/kg,IV).

FIG. 3 shows dose-dependent effects of a BMP6 antibody on serumbiomarkers of iron metabolism, Top: Serum hIgG concentration over timefollowing a single IV injection of Antibody 6 at the indicated doses.Bottom: Left panel is quantitative analysis of serum hepcidinconcentration after a single Antibody 6 or control human IgG injection,whereas right panel is serum iron concentration.

FIG. 4 shows therapeutic treatment of BMP6 Antibody in an ESA-resistantanemia of inflammation mouse model. Top: Experimental scheme ofBA-induced ESA-resistant anemia of inflammation model. Bottom:Erythropoiesis parameters at 13 days after BA treatment. HGB:hemoglobin; HCT: hematocrit; RETA: reticulocyte count; RET-HE:Reticulocyte hemoglobin equivalent. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001 versus BA+EPO+hIgG1.

FIG. 5 shows linear epitope mapping by HD×MS (hydrogen/deuteriumexchange coupled with mass spectometry). The epitope of BMP6 bound byAntibody 7 is shown (residues 88-102 of human BMP6 (QTLVHLMNPEYVPKP (SEQID NO: 98))). Using HD×MS, Antibody 676, a humanized version of acommercially available BMP6 antibody, was found to bind to an epitopeconsisting of residues 23-35 of human BMP6 (VSSASDYNSSELK (SEQ ID NO:99)).

FIG. 6 shows the protocol for Part 1 of the clinical program toinvestigate the safety and efficacy of BMP6 antibodies.

FIG. 7 shows the dose adjustment decision tree for the clinical programto investigate the safety and efficacy of BMP6 antibodies.

FIG. 8 shows the protocol for Part 2 of the clinical program toinvestigate the safety and efficacy of BMP6 antibodies.

FIG. 9 shows pharmacokinetics profiles of single dose Antibody 7 in malerats.

FIG. 10 shows dose-dependent effects of Antibody 7 on serum biomarkersof iron metabolism in rats. Shown is the quantitative analysis of serumhepcidin concentration after a single Antibody 7 or control (vehicle)injection at the indicated dose. Left panel shows an expanded view ofthe effects in the first 24 hours after administration.

FIG. 11 shows dependent effects of Antibody 7 on serum iron in rats.Shown is the quantitative analysis of serum iron concentration after asingle Antibody 7 or control (vehicle) injection at the indicated dose.Left panel shows an expanded view of the effects in the first 24 hoursafter administration.

FIG. 12 shows the concentration-time profile of single dose IV injectionof Antibody 7 (3mg/kg) in cynomolgus monkeys. Plotted is total Antibody7 concentration (both free and BMP6-bound).

FIG. 13 shows serum hepcidin and Fe concentration in male cynomogusmonkeys after a single intravenous injection of Antibody 7 at a dose of3 mg/kg. Data from three different monkeys is shown, together with themean.

FIG. 14 shows the peak TSAT (iron saturation, %) for hemodialysispatients before treatment (Baseline) and during the 3 days followingtreatment with 0.01 mg/kg Antibody 7 (Ab7) (3 Days Post Dose). Cohort 1patients all had pre-treatment Ferritin levels less than or equal to 500ng/mL. Cohort 2 patients all had pre-treatment Ferritin levels between500 and 1000 ng/mL. The bold line shows the median TSAT level for eachgroup.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating conditions associatedwith reduced iron, e.g., anemia, with inhibitors of BMP6.

BMP6 Inhibitors

A wide variety of BMP6 antagonists can be used in the methods of thepresent invention, such as, for example, antibodies, fusion proteins,adnectins, aptamers, anticalins, lipocalins, nucleic acids (e.g.,antisense molecules, such as RNA interfering agents and ribozymes),immunoconjugates (e.g., an antibody conjugated to a therapeutic agent),small molecules, fusion proteins, BMP6-derived peptidic compounds, andreceptor-based antagonists (e.g. soluble BMP6 receptor domains).

Nucleic Acids/Antisense Molecules

In another embodiment, the BMP6 antagonist employed in the methods ofthe present invention is an antisense nucleic acid molecule that iscomplementary to a gene encoding BMP6, or to a portion of that gene, ora recombinant expression vector encoding the antisense nucleic acidmolecule. As used herein, an “antisense” nucleic acid comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid can hydrogen bond to a sense nucleic acid.

The use of antisense nucleic acids to down-modulate the expression of aparticular protein in a cell is well known in the art (see e.g.,Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986; Askart, F. K. andMcDonnell, W. M. (1996) N. Eng. J. Med. 334:316-318; Bennett, M. R. andSchwartz, S. M. (1995) Circulation 92:1981-1993; Mercola, D. and Cohen,J. S. (1995) Cancer Gene Ther. 2:47-59; Rossi, J. J. (1995) Br. Med.Bull. 51:217-225; Wagner, R. W. (1994) Nature 372:333-335). An antisensenucleic acid molecule comprises a nucleotide sequence that iscomplementary to the coding strand of another nucleic acid molecule(e.g., an mRNA sequence) and accordingly is capable of hydrogen bondingto the coding strand of the other nucleic acid molecule. Antisensesequences complementary to a sequence of an mRNA can be complementary toa sequence found in the coding region of the mRNA, the 5′or3′untranslated region of the mRNA or a region bridging the coding regionand an untranslated region (e.g., at the junction of the 5′untranslatedregion and the coding region). Furthermore, an antisense nucleic acidcan be complementary in sequence to a regulators region of the geneencoding the mRNA, for instance a transcription initiation sequence orregulatory element. Preferably, an antisense nucleic acid is designed soas to be complementary to a region preceding or spanning the initiationcodon on the coding strand/or in the 3′ untranslated region of an mRNA.

Antisense nucleic acids can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of BMP6 mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of BMP6 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of BMP6 mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethypuracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminotnethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an amisense orientation , RNA transcribedfrom the inserted nucleic acid will be if an antisense orientation to atarget nucleic acid of interest, described further in the followingsubsection).

The antisense nucleic acid molecules that can be utilized in the methodsof the present invention are typically administered to a subject orgenerated in situ such that they hybridize with or bind to cellular mRNAand/or genomic DNA encoding BMP6 to thereby inhibit expression byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acid inmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using vectors well known in the art and described in, forexample, US20070111230 the entire contents of which are incorporatedherein. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule employedby the methods of the present invention can include an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

In another embodiment, an antisense nucleic acid used in the methods ofthe present invention is a compound that mediates RNAi. RNA interferingagents include, but are not limited to, nucleic acid molecules includingRNA molecules which are homologous to BMP6 or a fragment thereof, “shortinterfering RNA” (siRNA), “short hairpin” or “small hairpin RNA”(shRNA), and small molecules which interfere with or inhibit expressionof a target gene by RNA interference (RNAi). RNA interference is apost-transcriptional, targeted gene-silencing technique that usesdouble-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containingthe same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287,2431-2432 (2000); Zamore, P. D. et al. Cell 101, 25-33 (2000). Tuschl,T. et al. Genes Dev. 13, 3191-3197 (1999)). The process occurs when anendogenous ribonuclease cleaves the longer dsRNA into shorter, 21- or22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. Thesmaller RNA segments then mediate the degradation of the target mRNA.Kits for synthesis of RNAi are commercially available from, e.g., NewEngland Biolabs and Ambion. In one embodiment one or more of thechemistries described above for use in antisense RNA can be employed.

In still another embodiment, an antisense nucleic acid is a ribozyme.Ribozymes are catalytic RNA molecules with .ribonuclease activity whichare capable of cleaving a single-stranded nucleic acid, such as an mRNA,to which they have a complementary region. Thus, ribozymes (e.g.,hammerhead ribozymes (described in Haselhoff and Gerlach, 1988, Nature334:585-591) can be used to catalytically cleave BMP6 mRNA transcriptsto thereby inhibit translation of BMP6 mRNA.

Alternatively, gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of BMP6 (e.g., thepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the BMP6 gene. See generally, Helene, C., 1991,Anticancer Drug Des. 6(6):569-84; Helene. C. et al., 1992, Ann. N.Y.Acad. Sci. 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15.

Fusion Proteins and BMP6-Derived Peptidic Compounds

In another embodiment, the BMP6 antagonist used in the methods of thepresent invention is a fusion protein or peptidic compound derived fromthe BMP6 amino acid sequence. In particular, the inhibitory compoundcomprises a fusion protein or a portion of BMP6 (or a mimetic thereof)that mediates interaction of BMP6 with a target molecule such thatcontact of BMP6 with this fusion protein or peptidic compoundcompetitively inhibits the interaction of BMP6 with the target molecule.Such fusion proteins and peptidic compounds can be made using standardtechniques known in the art. For example, peptidic compounds can be madeby chemical synthesis using standard peptide synthesis techniques andthen introduced into cells by a variety of means known in the art forintroducing peptides into cells (e.g., liposome and the like).

The in vivo half-life of the fusion protein or peptidic compounds of theinvention can be improved by making peptide modifications, such as theaddition of N-linked glycosylation sites into the BMP6 peptidiccompound, or conjugating the peptidic BMP6 compound to poly(ethyleneglycol) (PEG; pegylation), e.g., via lysine-monopegylation. Suchtechniques have proven to be beneficial in prolonging the half-life oftherapeutic protein drugs. It is expected that pegylation of the BMP6polypeptides of the invention may result in similar pharmaceuticaladvantages.

In addition, pegylation can be achieved in any part of a polypeptide ofthe invention by the introduction of a nonnatural amino acid. Certainnonnatural amino acids can be introduced by the technology described inDeiters et al., J Am Chem Soc 125:11782-11783, 2003; Wang and Schultz,Science 301:964-967, 2003; Wang et al., Science 292:498-500, 2001; Zhanget al., Science 303:371-373, 2004 or in U.S. Pat. No. 7,083,970.Briefly, some of these expression systems involve site-directedmutagenesis to introduce a nonsense codon, such as an amber TAG, intothe open reading frame encoding a polypeptide of the invention. Suchexpression vectors are then introduced into a host that can utilize atRNA specific for the introduced nonsense codon and charged with thenonnatural amino acid of choice. Particular nonnatural amino acids thatare beneficial for purpose of conjugating moieties to the polypeptidesof the invention include those with acetylene and azido side chains. TheBMP6 polypeptides containing these novel amino acids can then bepegylated at these chosen sites in the protein.

Receptor-Based Antagonist

In another embodiment, the BMP6 antagonist used in the methods of thepresent invention is a receptor-based antagonist. Receptor-basedantagonists include soluble BMP6 receptors that bind BMP6 (or portionsthereof), respectively, and disrupt BMP6 activity and/or function. In aparticular embodiment, the receptor-based antagonist includes, but isnot limited to, soluble hemojuvelin or BMP Type I or Type II receptor.Versions of soluble hemojuvelin include those described in patentapplication publication US2010/0136015.

BMP6 Antibodies and Antigen-Binding Fragments Thereof

The present invention provides, as BMP6 inhibitors useful in the methodsand other embodiments and aspects of the invention described herein,antibodies and antigen-binding fragments thereof that specifically bindto human BMP6.

BMP6, a secreted BMP family growth factor ligand, has been identified asa critical endogenous regulator of hepatic expression of iron metabolismhormone hepcidin. Without being bound by any particular theory, thisdisclosure suggests a BMP6 antagonist antibody as a hepcidin-loweringtherapy is expected to benefit patients with iron-restricted anemia byovercoming resistance to Erythropoiesis Stimulating Agent (ESA), whichadds substantially to the morbidity of an underlying disease and isoften a predictor of adverse outcome.

Examples of such anti-human BMP6 antibodies are Antibodies 3, 5, 6 and7, whose sequences are listed in Table 1, and the anti-human BMP6antibodies described in Table 14.

Antibodies 5, 6 and 7 all bind with high affinity for human BMP6, withhigh selectivity over human BMP7, human BMP5 and human BMP2 (see FIG.1A). These antibodies also all demonstrate a decrease in serum hepcidinand an increase in serum iron in rats (see FIG. 2).

Additional examples of anti-human BMP6 antibodies are LY3113593 (see,e.g., clinical trial NCT02604160). Additional examples of anti-humanBMP6 antibodies are described in PCT application publication numberWO2014/099391, the contents of which are hereby incorporated byreference in their entirety. Particular anti-human BMP6 antibodies ofWO2014/099391 include the antibodies described in Table 14.

TABLE 14 Examples of anti-human BMP6 antibodies SEQ ID Sequence NameSequence NO: Antibody 1 LCDR1 RSSENIYRNLA 1 LCDR2 AATNLAD 2 LCDR3QGIWGTPLT 3 Light Chain DIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWY 4Variable Region QQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDF (aa)TLTISSLQPEDFATYYCQGIWGTPLTFGGGIKVEIK Light Chain (aa)DIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWY 5QQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQGIWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC HCDR1 GYTFTSYAMH 6 HCDR2YINPYNDGTKYNENFK 7 HCDR3 RPFGNAMDI 8 Heavy ChainQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAM 92 Variable RegionHWVRQAPGQGLEWMGYINPYNDGTKYNENFKGR (aa)VTITADESTSTAYMELSSLRSEDTAVYYCARRPFG NAMDIWGQGTLVTVSS Heavy Chain (aa)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAM 93 HWVRQAPGQGLEWMGYINPYNDGTKYNENFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARRPFG NAMDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Antibody 2 LCDR1 RSSENIYRNLA 1 LCDR2AATNLAD 2 LCDR3 QGIWGTPLT 3 Light ChainDIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWY 4 Variable RegionQQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDF (aa)TLTISSLQPEDFATYYCQGIWGTPLTFGGGTKVEIK Light Chain (aa)DIQMTQSPSSLSASVGDRVTITCRSSENIYRNLAWY 5QQKPGKAPKLLIYAATNLADGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQGIWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC HCDR1 GYTFTSYAMH 6 HCDR2YINPYNRGTKYNENFK 94 HCDR3 RPFGNAMDI Heavy ChainQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAM 95 Variable RegionHWVRQAPGQGLEWMGYINPYNRGTKYNENFKGR (aa)VTITADESTSTAYMELSSLRSEDTAVYYCARRPFG NAMDIWGQGTLVTVSS Heavy Chain (aa)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAM 96 HWVRQAPGQGLEWMGYINPYNRGTKYNENFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARRPFG NAMDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

To provide further evidence that targeting this pathway can conferimprovement of functional endpoints, we tested the ability ofBMP6-specific antibodies, Antibodies 5 to 7, to modulate serumbiomarkers for iron metabolism in normal mice and rats, and to reverseESA-resistant anemia in a mouse model of anemia of inflammation. Wefound that a single injection of animals with BMP6 antibody resulted ina sustained increase of serum iron levels, accompanied by potentsuppression of circulating hepcidin. Furthermore, therapeutic treatmentof mice subjected to inflammation-induced anemia significantly improvederythropoietic parameters in response to concurrent erythropoietintreatment.

In this disclosure, inhibition of BMP6 signaling in a mouse model ofanemia of inflammation substantially improved iron-dependent red cellparameters.

The BMP6 antagonist antibodies disclosed herein represent a noveltherapeutic approach to safely improve anemia with erythropoietinhypo-responsiveness. Without being bound by any particular theory, thisdisclosure suggests that this may occur through mobilization andavailability of iron store to the demand from erythroid compartment.

In one embodiment, the present invention provides isolated antibodies orantigen-binding fragments thereof that bind with a 100-, 500- or1000-fold higher affinity for human BMP6 protein, than to any of: humanBMP5 or human BMP7 protein. Specificity to BMP6 without binding to BMP7is important, as knock-out of BMP6 is not lethal to mice. However,knock-out mice for BMP7 die after birth with kidney, eye and bonedefects. Individual knock-outs of either gene do not altercardiogenesis, but a double knock-out of BMP6 and BMP7 demonstratedseveral defects and delays in the heart; embryos died to cardiacinsufficiency. BMP7 is important in preventing progression of chronicheart disease associated with fibrosis. Therefore, cross-reactivity ofan anti-BMP6 antibody with BMP7 is not desirable. Antibodies providedherein are specific to BMP6 over BMP7; See, for example, Table 4A. FIG.1B also shows evidence for binding specificity to human BMP6 over humanBMP2, BMP5 and BMP7 proteins. In contrast, a commercially-available BMP6antibody from R&D Systems, for example, was revealed to have strongcross-reactivity to BMP7 in a reporter gene assay, and to inhibit bothBMP6 and BMP7.

Antibodies of the invention include, but are not limited to, the humanmonoclonal antibodies, isolated as described, in the Examples (seeSection 6 below). Examples of such anti-human BMP6 antibodies areAntibodies 3, 5, 6 and 7, whose sequences are listed in Table 1.

Matured antibody 7 is derived from NOV0442_VL(YGQ) Germlining/PTMremoval, which is derived from parental IgG hit NOV0442 (VH3_3-15,V11_1e). Antibody 7 binds with high affinity for human BMP6 in an ELISAbinding assay, with selectivity over human BMP7 of over 500-fold (i.e.,an affinity to human BMP6 over 500-fold greater than to human BMP7).This antibody also has no detectable activity against human BMP2 orBMP5. The BMP6 peptide recognized by parental IgG NOV0442 and Antibody 7is shown in FIG. 5. The peptide comprises amino acids QTLVHLMNPEYVPKP(SEQ ID NO: 98) of human BMP6. In contrast to IgG NOV0442 and Antibody7, humanized mAb507 (R&D Systems) binds to the sequence VSSASDYNSSELK(SEQ ID NO: 99) of human BMP6. Thus, the epitope recognized by IgGNOV0442 and Antibody 7 represents a novel BMP6 epitope. Antibody 7 alsoinhibits BMP6 binding to receptors in vitro. Binding of BMP6 to BMPR1Ais inhibited maximally 59%; binding to BMPR1B is inhibited maximally85%; and binding to RGM-c is inhibited maximally 72%. A single 10 mg/kgtreatment in rats led to sustained suppression of circulating hepcidin.The estimated minimum effective dose in mice is less than or equal to0.1 mg/kg. Serum iron also showed an increase, and hepcidin showed adecrease after a single Antibody dose in monkeys of 3 mg/kg. In mice,wherein Brucella abortus antigen was used to simulate anemia, thetreatment effect of Antibody 7 (2 mg/kg) is consistent with clinicallysignificant erythropoietic response to chronic EPO therapy, with gradualhemoglobin increase of >2.0 g/dL from baseline.

In antibody 7, a potential post-translational modification site wasremoved by an N51Q mutation within LCDR2 to increase later producthomogeneity. The antibody derived from the VH3/lambdal framework wasengineered in order to match the closest human germline sequence: in VHby a V40A mutation, in VL by D1Q, I2S mutations and introduction ofamino acids Y49 and G50 to repair the framework in which these 2residues were initially missing.

This work resulted in Antibody 7 (=NOV0958=NOV0806_VH[V40A]_VL[D1Q, I2S,Y49, G50, N51Q]).

Antibodies 3, 5, 6 and 7 all show high specificity for human BMP6protein compared to human BMP2, BMP5 or BMP7 protein. The epitope of allthese antibodies is predicted to be the same as they are all derivedfrom a single parental Fab before affinity maturation. Antibody 3, forexample, shares the same Fab clone with both antibodies 5 and 7 beforeaffinity maturation of HCDR2. Antibody 5 is derived from NOV0442(VH3_3-15, VI1_1e)→NOV0442_VL(YGQ)→(HCDR2 affinity maturation)→Antibody5. Antibody 3 is derived from NOV0442 (VH3_3-15,VI1_1e)→NOV0442_VL(YGS)→(HCDR2 affinity maturation)→Antibody 3.Additional details regarding the generation of the antibodies describedherein are provided in the Examples.

The present invention provides antibodies that specifically bind BMP6(e.g., human BMP6 protein), said antibodies comprising a VH domainlisted in Table 1 and/or Table 14. The present invention also providesantibodies that specifically bind to BMP6 protein, said antibodiescomprising a VH CDR having an amino acid sequence of any one of the VHCDRs listed in Table 1 and/or Table 14. In particular, the inventionprovides antibodies that specifically bind to BMP6 protein, saidantibodies comprising (or alternatively, consisting of) one, two, three,four, five or more VH CDRs having an amino acid sequence of any of theVH CDRs listed in Table 1 and/or Table 14.

The invention also provides antibodies and antigen-binding fragmentsthereof that specifically bind to BMP6, said antibodies orantigen-binding fragments thereof comprising (or alternatively,consisting of) a amino acid sequence listed in Table 1 and/or Table 14,wherein no more than about 10 amino acids in a framework sequence (forexample, a sequence which is not a CDR) have been mutated (wherein amutation is, as various non-limiting examples, an addition, substitutionor deletion). The invention also provides antibodies and antigen-bindingfragments thereof that specifically bind to BMP6, said antibodies orantigen-binding fragments thereof comprising alternatively, consistingof) a VH amino acid sequence listed in Table 1 and/or Table 14, whereinno more than 10 amino acids in a framework sequence (for example, asequence which is not a CDR) have been mutated (wherein a mutation is,as various non-limiting examples, an addition, substitution ordeletion).

The invention also provides antibodies and antigen-binding fragmentsthereof that specifically bind to BMP6, said antibodies orantigen-binding fragments thereof comprising (or alternatively,consisting of) a VH amino acid sequence listed in Table 1 and/or Table14, wherein no more than about 20 amino acids in a framework sequence(for example, a sequence which is not a CDR) have been mutated (whereina mutation is, as various non-limiting examples, an addition,substitution or deletion). The invention also provides antibodies andantigen-binding fragments thereof that specifically bind to BMP6, saidantibodies or antigen-binding fragments thereof comprising (oralternatively, consisting of) a VH amino acid sequence listed in Table 1and/or Table 14, wherein no more than 20 amino acids in a frameworksequence (for example, a sequence which is not a CDR) have been mutated(wherein a mutation is, as various non-limiting examples, an addition,substitution or deletion).

The invention also provides antibodies and antigen-binding fragmentsthereof that specifically bind to BMP6, said antibodies orantigen-binding fragments thereof comprising (or alternatively,consisting of) a VL amino acid sequence listed in Table 1 and/or Table14, wherein no more than about 10 amino acids in a framework sequence(for example, a sequence which is not a CDR) have been mutated (whereina mutation is, as various non-limiting examples, an addition,substitution or deletion). The invention also provides antibodies andantigen-binding fragments thereof that specifically bind to BMP6, saidantibodies or antigen-binding fragments thereof comprising (oralternatively, consisting of) a VL amino acid sequence listed in Table 1and/or Table 14, wherein no more than 10 amino acids in a frameworksequence (for example, a sequence which is not a CDR) have been mutated(wherein a mutation is, as various non-limiting examples, an addition,substitution or deletion).

The invention also provides antibodies and antigen-binding fragmentsthereof that specifically bind to BMP6, said antibodies orantigen-binding fragments thereof comprising (or alternatively,consisting of) a VL amino acid sequence listed in Table 1 and/or Table14, wherein no more than about 20 amino acids in a framework sequence(for example, a sequence which is not a CDR) have been mutated (whereina mutation is, as various non-limiting examples, an addition,substitution or deletion). The invention also provides antibodies andantigen-binding fragments thereof that specifically bind to BMP6, saidantibodies or antigen-binding fragments thereof comprising (oralternatively, consisting of) a VL amino acid sequence listed in Table 1and/or Table 14, wherein no more than 20 amino acids in a frameworksequence (for example, a sequence which is not a CDR) have been mutated(wherein a mutation is, as various non-limiting examples, an addition,substitution or deletion).

The present invention provides antibodies and antigen-binding fragmentsthereof that specifically bind to BMP6 protein, said antibodies orantigen-binding fragments thereof comprising a VL domain listed in Table1 and/or Table 14. The present invention also provides antibodies andantigen-binding fragments thereof that specifically bind to BMP6protein, said antibodies or antigen-binding fragments thereof comprisinga VL CDR having an amino acid sequence of any one of the VL CDRs listedin Table 1 and/or Table 14. In particular, the invention providesantibodies and antigen-binding fragments thereof that specifically bindto BMP6 protein, said antibodies or antigen-binding fragments thereofcomprising (or alternatively, consisting of) one, two, three or more VLCDRs having an amino acid sequence of any of the VL CDRs listed in Table1 and/or Table 14.

Other antibodies and antigen-binding fragments thereof of the inventioninclude amino acids that have been mutated, yet have at least 60, 70,80, 90 or 95 percent identity in the CDR regions with the CDR regionsdepicted in the sequences described in Table 1 and/or Table 14, In oneembodiment, it includes mutant amino acid sequences wherein no more than1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions whencompared with the CDR regions depicted in the sequence described inTable 1 and/or Table 14.

The present invention also provides nucleic acid sequences that encodeVH, VL, the full length heavy chain, and the full length light chain ofthe antibodies and antigen-binding fragments thereof that specificallybind to BMP6 protein. Such nucleic acid sequences can be optimized forexpression in mammalian cells (for example. Table 1 shows examplenucleic acid sequences for the heavy chain and light chain of Antibodies3, 5, 6 and 7).

TABLE 1 Examples of BMP6 Antibodies of the Present Invention SEQ ID NO:ANTIBODY 3 Kabat HCDR1 SYVVH 9 Kabat HCDR2 RIKDHKQGYTTAYAASVKG 10 KabatHCDR3 VERSKSGFDN 11 Chothia HCDR1 GFTFSSY 12 Chothia HCDR2 KDHKQGYT 13Chothia HCDR3 VERSKSGFDN 14 VH QVQLVESGGGLVKPGGSLRLSCAASGFT 15FSSYVVHWVRQAPGKGLEWVGRIKDHK QGYTTAYAASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARVERSKSGFDN WGQGTLVTVSS DNA VHCAGGTGCAATTGGTGGAAAGCGGCGGT 16 GGCCTGGTGAAACCAGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCTCCGGATTC ACCTTTTCTTCTTACGTTGTTCATTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCG AGTGGGTGGGCCGTATCAAAGACCACAAACAGGGCTACACTACTGCTTATGCCG CCTCTGTGAAAGGCCGCTTTACCATTAGCCGCGATGATTCGAAAAACACCCTGTA TCTGCAAATGAACAGCCTGAAAACCGAAGATACGGCCGTGTATTATTGCGCGCG TGTTGAACGTTCTAAATCTGGTTTCGATAACTGGGGCCAAGGCACCCTGGTGACT GTTAGCTCA Heavy ChainQVQLVESGGGLVKPGGSLRLSCAASGFT 17 FSSYVVHWVRQAPGKGLEWVGRIKDHKQGYTTAYAASVKGRFTISRDDSKNTLYL QMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK DNA HeavyCAGGTGCAATTGGTGGAAAGCGGCGGT 18 Chain GGCCTGGTGAAACCAGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCTCCGGATTC ACCTTTTCTTCTTACGTTGTTCATTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCG AGTGGGTGGGCCGTATCAAAGACCACAAACAGGGCTACACTACTGCTTATGCCG CCTCTGTGAAAGGCCGCTTTACCATTAGCCGCGATGATTCGAAAAACACCCTGTA TCTGCAAATGAACAGCCTGAAAACCGAAGATACGGCCGTGTATTATTGCGCGCG TGTTGAACGTTCTAAATCTGGTTTCGATAACTGGGGCCAAGGCACCCTGGTGACT GTTAGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCA AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACA CCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA GCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCT TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA AGAGCCTCTCCCTGTCTCCGGGTAAA Kabat LCDR1TGSSSNIGAGYSVH 19 Kabat LCDR2 GSSERPS 20 Kabat LCDR3 QSWDSSQTLVV 21Chothia LCDR1 SSSNIGAGYS 22 Chothia LCDR2 GSS 23 Chothia LCDR3 WDSSQTLV24 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIG 25 AGYSVHWYQQLPGTAPKLLIYGSSERPSGVPDRFSGSKSGTSASLAITGLQAEDEAD YYCQSWDSSQTLVVFGGGTKLTVL DNA VLCAGAGCGTGCTGACCCAGCCGCCGAGC 26 GTGAGCGGTGCACCGGGCCAGCGCGTGACCATTAGCTGTACCGGCAGCAGCAGC AACATTGGTGCTGGTTACTCTGTGCATTGGTACCAGCAGCTGCCGGGCACGGCGC CGAAACTGCTGATCTATGGTAGCTCTGAACGCCCGAGCGGCGTGCCGGATCGCT TTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAG CAGAAGACGAAGCGGATTATTACTGCCAGTCTTGGGACTCTTCTCAGACTCTGGT TGTGTTTGGCGGCGGCACGAAGTTAAC CGTCCTALight Chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIG 27AGYSVHWYQQLPGTAPKLLIYGSSERPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPK AAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS DNA Light CAGAGCGTGCTGACCCAGCCGCCGAGC 28 ChainGTGAGCGGTGCACCGGGCCAGCGCGTG ACCATTAGCTGTACCGGCAGCAGCAGCAACATTGGTGCTGGTTACTCTGTGCATT GGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTATGGTAGCTCTG AACGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCG CCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCC AGTCTTGGGACTCTTCTCAGACTCTGGTTGTGTTTGGCGGCGGCACGAAGTTAAC CGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCT GAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACC CGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAG TGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCT ATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGG TCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA ANTIBODY 5 Kabat HCDR1 SYVVH 29 Kabat HCDR2RIKRESSSYTTMYAAPVKG 30 Kabat HCDR3 VERSKSGFDN 31 Chothia HCDR1 GFTFSSY32 Chothia HCDR2 KRESSSYT 33 Chothia HCDR3 VERSKSGFDN 34 VHQVQLVESGGGLVKPGGSLRLSCAASGFT 35 FSSYVVHWVRQAPGKGLEWVGRIKRESSSYTTMYAAPVKGRFTISRDDSKNTLYLQ MNSLKTEDTAVYYCARVERSKSGFDNW GQGTLVTVSSDNA VH CAGGTGCAGCTGGTGGAATCAGGCGGC 36 GGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTC ACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGG AGTGGGTCGGACGGATTAAGAGAGAGTCCTCTAGCTACACTACTATGTACGCCGC TCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTAC CTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGA GTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACC GTGTCTAGC Heavy ChainQVQLVESGGGLVKPGGSLRLSCAASGFT 37 FSSYVVHWVRQAPGKGLEWVGRIKRESSSYTTMYAAPVKGRFTISRDDSKNTLYLQ MNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK DNA HeavyCAGGTGCAGCTGGTGGAATCAGGCGGC 38 Chain GGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTC ACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGG AGTGGGTCGGACGGATTAAGAGAGAGTCCTCTAGCTACACTACTATGTACGCCGC TCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTAC CTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAGA GTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCACC GTGTCTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGC AAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTC CCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCAC ACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACA AGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCG TGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGA GGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGG AGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGG ACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAG CCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGG TGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGA CCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCA ACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGG GCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCC AGAAGTCCCTGAGCCTGAGCCCCGGCA AG Kabat LCDR1TGSSSNIGAGYSVH 39 Kabat LCDR2 GQSERPS 40 Kabat LCDR3 QSWDSSQTLVV 41Chothia LCDR1 SSSNIGAGYS 42 Chothia LCDR2 GQS 43 Chothia LCDR3 WDSSQTLV44 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIG 45 AGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEAD YYCQSWDSSQTLVVFGGGTKLTVL DNA VLCAGTCAGTCCTGACTCAGCCCCCTAGC 46 GTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTA ATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCC CTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGG CTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGG TGGTGTTCGGCGGAGGCACTAAGCTGA CCGTGCTGLight Chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIG 47AGYSVHWYQQLPGTAPKLLIYGQSERPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPK AAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS DNA Light CAGTCAGTCCTGACTCAGCCCCCTAGC 48 ChainGTCAGCGGCGCTCCCGGTCAGAGAGTG ACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACT GGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAG AGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCG CTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTC AGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGA CCGTGCTGGGTCAGCCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCA GCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCT ACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCG GCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCA GCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCC AGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCA GC ANTIBODY 6 Kabat HCDR1 SYVVH 49 KabatHCDR2 RTRHSDMGYATSYAAPVKG 50 Kabat HCDR3 VERSKSGFDN 51 Chothia HCDR1GFTFSSY 52 Chothia HCDR2 RHSDMGYA 53 Chothia HCDR3 VERSKSGFDN 54 VHQVQLVESGGGLVKPGGSLRLSCAASGFT 55 FSSYVVHWVRQAPGKGLEWVGRTRHSDMGYATSYAAPVKGRFTISRDDSKNTLYL QMNSLKTEDTAVYYCARVERSKSGFDN WGQGTLVTVSSDNA VH CAGGTGCAGCTGGTGGAATCAGGCGGC 56 GGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTC ACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGG AGTGGGTCGGACGGACTAGACACTCAGATATGGGCTACGCTACTAGCTACGCCG CTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTA CCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAG AGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCAC CGTGTCTAGC Heavy ChainQVQLVESGGGLVKPGGSLRLSCAASGFT 57 FSSYVVHWVRQAPGKGLEWVGRTRHSDMGYATSYAAPVKGRFTISRDDSKNTLYL QMNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK DNA HeavyCAGGTGCAGCTGGTGGAATCAGGCGGC 58 Chain GGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTC ACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGG AGTGGGTCGGACGGACTAGACACTCAGATATGGGCTACGCTACTAGCTACGCCG CTCCCGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGTA CCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTAG AGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCAC CGTGTCTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAG CAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTT CCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCAC ACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACA AGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCG TGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGA GGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGG AGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGG ACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAG CCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGG TGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGA CCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCA ACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACG GCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGG GCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCC AGAAGTCCCTGAGCCTGAGCCCCGGCA AG Kabat LCDR1TGSSSNIGAGYSVH 59 Kabat LCDR2 GQSERPS 60 Kabat LCDR3 QSWDSSQTLVV 61Chothia LCDRI SSSNIGAGYS 62 Chothia LCDR2 GQS 63 Chothia LCDR3 WDSSQTLV64 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIG 65 AGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEAD YYCQSWDSSQTLVVFGGGTKLTVL DNA VLCAGTCAGTCCTGACTCAGCCCCCTAGC 66 GTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTA ATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCC CTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGG CTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGG TGGTGTTCGGCGGAGGCACTAAGCTGA CCGTGCTGLight Chain QSVLTQPPSVSGAPGQRVITSCTGSSSNIG 67AGYSVHWYQQLPGTAPKLLIYGQSERPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPK AAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS DNA Light CAGTCAGTCCTGACTCAGCCCCCTAGC 68 ChainGTCAGCGGCGCTCCCGGTCAGAGAGTG ACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACT GGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAG AGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCG CTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTC AGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGA CCGTGCTGGGTCAGCCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCA GCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCT ACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCG GCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCA GCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCC AGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCA GC ANTIBODY 7 Kabat HCDR1 SYVVH 69 KabatHCDR2 RIRLETHGYAAEYAASVKG 70 Kabat HCDR3 VERSKSGFDN 71 Chothia HCDR1GFTFSSY 72 Chothia HCDR2 RLETHGYA 73 Chothia HCDR3 VERSKSGFDN 74 VHQVQLVESGGGLVKPGGSLRLSCAASGFT 75 FSSYVVHWVRQAPGKGLEWVGRIRLETHGYAAEYAASVKGRFTISRDDSKNTLYLQ MNSLKTEDTAVYYCARVERSKSGFDNW GQGTLVTVSSDNA VH CAGGTGCAGCTGGTGGAATCAGGCGGC 76 GGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGTTC ACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGG AGTGGGTCGGACGGATTAGACTGGAAACTCACGGCTACGCCGCCGAGTACGCCG CTAGTGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGT ACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTA GAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCA CCGTGTCTAGC Heavy ChainQVQLVESGGGLVKPGGSLRLSCAASGFT 77 FSSYVVHWVRQAPGKGLEWVGRIRLETHGYAAEYAASVKGRFTISRDDSKNTLYLQ MNSLKTEDTAVYYCARVERSKSGFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK DNA HeavyCAGGTGCAGCTGGTGGAATCAGGCGGC 78 Chain GGACTGGTCAAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTC ACCTTCTCTAGCTACGTGGTGCACTGGGTCAGACAGGCCCCTGGTAAAGGCCTGG AGTGGGTCGGACGGATTAGACTGGAAACTCACGGCTACGCCGCCGAGTACGCCG CTAGTGTGAAGGGCCGGTTCACTATCTCTAGGGACGACTCTAAGAACACCCTGT ACCTGCAGATGAATAGCCTGAAAACCGAGGACACCGCCGTCTACTACTGCGCTA GAGTGGAACGGTCTAAGTCAGGCTTCGATAACTGGGGTCAGGGCACCCTGGTCA CCGTGTCTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCA GCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACT TCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGC ACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGG TGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACC ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGAC AAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCC GTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCG AGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAG GAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAG GACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCA GCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAG GTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGC AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGAC GGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAG GGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGTCCCTGAGCCTGAGCCCCGGC AAG Kabat LCDR1TGSSSNIGAGYSVH 79 Kabat LCDR2 GQSERPS 80 Kabat LCDR3 QSWDSSQTLVV 81Chothia LCDR1 SSSNIGAGYS 82 Chothia LCDR2 GQS 83 Chothia LCDR3 WDSSQTLV84 VL QSVLTQPPSVSGAPGQRVTISCTGSSSNIG 85 AGYSVHWYQQLPGTAPKLLIYGQSERPSGVPDRFSGSKSGTSASLAITGLQAEDEAD YYCQSWDSSQTLVVFGGGTKLTVL DNA VLCAGTCAGTCCTGACTCAGCCCCCTAGC 86 GTCAGCGGCGCTCCCGGTCAGAGAGTGACTATTAGCTGCACCGGCTCTAGCTCTA ATATCGGCGCTGGCTATAGCGTGCACTGGTATCAGCAGCTGCCCGGCACCGCCC CTAAGCTGCTGATCTACGGTCAGTCAGAGCGGCCTAGCGGCGTGCCCGATAGGT TTAGCGGCTCTAAGTCAGGCACTAGCGCTAGTCTGGCTATCACCGGCCTGCAGG CTGAGGACGAGGCCGACTACTACTGTCAGTCCTGGGACTCTAGTCAGACCCTGG TGGTGTTCGGCGGAGGCACTAAGCTGA CCGTGCTGLight Chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIG 87AGYSVHWYQQLPGTAPKLLIYGQSERPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSWDSSQTLVVFGGGTKLTVLGQPK AAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS DNA Light CAGTCAGTCCTGACTCAGCCCCCTAGC 88 ChainGTCAGCGGCGCTCCCGGTCAGAGAGTG ACTATTAGCTGCACCGGCTCTAGCTCTAATATCGGCGCTGGCTATAGCGTGCACT GGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTACGGTCAGTCAG AGCGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGCTCTAAGTCAGGCACTAGCG CTAGTCTGGCTATCACCGGCCTGCAGGCTGAGGACGAGGCCGACTACTACTGTC AGTCCTGGGACTCTAGTCAGACCCTGGTGGTGTTCGGCGGAGGCACTAAGCTGA CCGTGCTGGGTCAGCCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCA GCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCT ACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCG GCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCA GCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCC AGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCA GC

Other antibodies and antigen-binding fragments thereof of the inventioninclude those wherein the amino acids or nucleic acids encoding theamino acids have been mutated, yet have at least 60, 70, 80, 90 or 95percent identity to the sequences described in Table 1 and/or Table 14.In one embodiment, it include mutant amino acid sequences wherein nomore than 1, 2, 3, 4 or 5 amino acids have been mutated in the variableregions when compared with the variable regions depicted in the sequencedescribed in Table 1 and/or Table 14, while retaining substantially thesame therapeutic activity.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:9, 10 and 11, respectively, and the LCDR1, LCDR2, and LCDR3 sequences ofSEQ ID NOs: 19, 20 and 21, respectively.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:12, 13 and 14, respectively, and the LCDR1, LCDR2, and LCDR3 sequencesof SEQ ID NOs: 22, 23 and 24, respectively.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:29, 30 and 31, respectively, and the LCDR1, LCDR2, and LCDR3 sequencesof SEQ ID NOs: 39, 40 and 41, respectively.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:32, 33 and 34, respectively, and the LCDR1, LCDR2, and LCDR3 sequencesof SEQ ID NOs: 42, 43 and 44, respectively.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:49, 50 and 51, respectively, and the LCDR1, LCDR2, and LCDR3 sequencesof SEQ ID NOs: 59, 60 and 61, respectively.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:52, 53 and 54, respectively, and the LCDR1, LCDR2, and LCDR3 sequencesof SEQ ID NOs: 62, 63 and 64, respectively.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:69, 70 and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequencesof SEQ ID NOs: 79, 80 and 81, respectively.

In another specific embodiment, the present invention provides anisolated antibody or antigen-binding fragment thereof, which binds humanBMP6 and comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ NOs: 72,73 and 74, respectively, and the LCDR1, LCDR2, and LCDR3 sequences ofSEQ ID NOs: 82, 83 and 84, respectively.

Since each of these antibodies can bind to BMP6, the VH, VL, full lengthlight chain, and full length heavy chain sequences (amino acid sequencesand the nucleotide sequences encoding the amino acid sequences) can be“mixed and matched” to create other BMP6-binding antibodies andantigen-binding fragments thereof of the invention. Such “mixed andmatched” BMP6-binding antibodies can be tested using the binding assaysknown in the art (e.g., ELISAs, and other assays described in theExample section). When these chains are mixed and matched, a VH sequencefrom a particular VH/VL pairing should be replaced with a structurallysimilar VH sequence. Likewise a full length heavy chain sequence from aparticular full length heavy chain/full length light chain pairingshould be replaced with a structurally similar full length heavy chainsequence. Likewise, a VL sequence from a particular VH/VL pairing shouldbe replaced with a structurally similar VL sequence. Likewise a fulllength light chain sequence from a particular full length heavychain/full length light chain pairing should be replaced with astructurally similar full length light chain sequence.

In another aspect, the present invention provides BMP6-bindingantibodies that comprise the heavy chain and light chain CDR1s, CDR2sand CDR3s as described in Table 1 and/or Table 14, or combinationsthereof. The CDR regions are delineated using the Kabat system (Kabat etal. 1991 Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242), or using the Chothia system [Chothia et al. 1987 J. Mol. Biol.196: 901-917: and Al-Lazikani et al. 1997 J. Mol. Biol. 273: 927-948].

Given that each of these antibodies can bind to BMP6 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequencescan be “mixed and matched” (i.e., CDRs from different antibodies can bemixed and match, although each antibody must contain a VH CDR1, 2 and 3and a VL CDR1, 2 and 3 to create other BMP6-binding binding molecules ofthe invention, Such “mixed and matched” BMP6-binding antibodies can betested using the binding assays known in the art and those described inthe Examples (e.g., ELISAs). When VH CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular VHsequence should be replaced with a structurally similar CDR sequence(s).Likewise, when VL CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular VL sequence should be replacedwith a structurally similar CDR sequence(s). It will be readily apparentto the ordinarily skilled artisan that novel VH and VL sequences can becreated by mutating one or more VH and/or VL CDR region sequences withstructurally similar sequences from the CDR sequences shown herein formonoclonal antibodies of the present invention.

Accordingly, the present invention provides an isolated monoclonalantibody or antigen binding region thereof comprising a heavy chainvariable region CDR1 comprising an amino acid sequence selected from anyof SEQ ID NOs: 29, 49, 69, 12, 32, 52, 72, or 9; a heavy chain variableregion CDR2 comprising an amino acid sequence selected from any of SEQID NOs: 10, 30, 50, 70, 13, 33, 53, or 73; a heavy chain variable regionCDR3 comprising an amino acid sequence selected from any of SEQ ID NOs:11, 31, 51, 71, 14, 34, 54, or 74; a light chain variable region CDR1comprising an amino acid sequence selected from any of SEQ ID NOs: 19,39, 59, 79, 22, 42, 62, or 82; a light chain variable region CDR2comprising an amino acid sequence selected from any of SEQ ID NOs: 20,40, 60, 80, 23, 43, 63, or 83; and a light chain variable region CDR3comprising an amino acid sequence selected from any of SEQ ID NOs: 21,41, 61, 81, 24, 44, 64, or 84; wherein the antibody specifically bindsBMP6.

In one embodiment, an antibody that specifically binds to BMP6 is anantibody that is described in Table 1 and/or Table 14.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are the product of or“derived from” a particular germline sequence if the variable regions orfull length chains of the antibody are obtained from a system that useshuman germline immunoglobulin genes. Such systems include immunizing atransgenic mouse carrying human immunoglobulin genes with the antigen ofinterest or screening a human immunoglobulin gene library displayed onphage with the antigen of interest. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutations. However, in the VH or VLframework regions, a selected human antibody typically is at least 90%identical in amino acids sequence to an amino acid sequence encoded by ahuman germline immunoglobulin gene and contains amino acid residues thatidentify the human antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or99% identical in amino acid sequence to the amino acid sequence encodedby the germline immunoglobulin gene. Typically, a recombinant humanantibody will display no more than 10 amino acid differences from theamino acid sequence encoded by the human germline immunoglobulin gene inthe VH or VL framework regions. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

BMP Family Members and Hepcidin

In one embodiment, the invention provides an antibody or bindingfragment thereof that specifically binds to BMP6 is an antibody. In oneembodiment, the antibody or binding fragment thereof is described inTable 1 and/or Table 14. In one embodiment, the antibody or bindingfragment thereof specifically binds to BMP6 but not to other BMPproteins (such as BMP2, BMP5 or BMP7). BMP6, a secreted BMP familygrowth factor ligand, is a 30 kDa disulfide-linked homodimer in itsmature active form. The protein is a member of the TGF-Beta superfamily.Bone morphogenetic proteins are known for their ability to induce thegrowth of bone and cartilage. BMP6 is able to induce all osteogenicmarkers in mesenchymal stem cells.

The bone morphogenetic proteins (BMPs) are a family of secretedsignaling molecules that can induce ectopic bone growth. BMPs are partof the transforming growth factor-beta (TGF-Beta) superfamily. BMPs wereoriginally identified by an ability of demineralized bone extract toinduce endochondral osteogenesis in vivo in an extraskeletal site. Basedon its expression early in embryogenesis, the BMP encoded by this genehas a proposed role in early development. In addition, the fact thatthis BMP is closely related to BMP5 and BMP7 has led to speculation ofpossible bone inductive activity. An additional function of BMP6 hasbeen identified as described in Nature Genetics April; 41 [4]:386-8.

Mice with a knock-out of BMP6 are viable and fertile, and show normalbone and cartilage development.

BMP6 is the key regulator of hepcidin, the small peptide secreted by theliver which is the major regulator of iron metabolism in mammals.Hepcidin controls both the amount of dietary iron absorbed in theduodenum and iron released by reticuloendothelial cells. Hepcidin isupregulated by a variety of stimuli, including inflammation and ironoverload, and downregulated by anemia, hypoxia, and iron deficiency.

Without being bound by any particular theory, this disclosure suggeststhat a BMP6 antagonist antibody as a hepcidin-lowering therapy isexpected to benefit patients with iron-restricted anemia by overcomingresistance to Erythropoiesis Stimulating Agent (ESA), which addssubstantially to the morbidity of an underlying disease and is often apredictor of adverse outcome. Through its interaction with BMPR1 andBMPR2 receptors, it induces receptors dimerization and transcription ofhepcidin. BMP6 also binds to HJV co-receptor in liver and muscle cells.

Thus, BMP6 is known to increase expression of hepcidin. Hepcidin isknown to be a key hormone involved in iron homeostasis. High hepcidinlevels are associated with iron restricted erythropoiesis in ACD.

WO 2010/056981 disclosed that administration to mice of an antibody toBMP6 decreased hepcidin and increased iron.

BMP6 is further described in the art, e.g.: Hahn et al. 1992 Genomics14: 759-62; Sauermann et al. 1993 J. Neurosci. Res. 33: 142; Celeste etal. 1991 Proc. Natl. Acad. Sci. USA 87: 9843; Schluesener et al. 1995Atherosclerosis 113: 153; Gitelman et al. 1994 J. Cell Biol. 126: 1595;Barnes et al. 1997 W. J. Urol. 13: 337; and Hamdy et al. 1997 CancerRes. 57: 4427.

BMP2, like other bone morphogenetic proteins, plays an important role inthe development of bone and cartilage. It is involved in the hedgehogpathway, TGF-Beta signaling pathway, and in cytokine-cytokine receptorinteraction. It is also involved in cardiac cell differentiation andepithelial to mesenchymal transition. BMP2 has many essential roles, asnoted by Kishimoto et al. 1997 Dev. 124: 4457; Ma et al. 2005 Dev. 132:5601; Wang et al. Bone 48: 524; and Rosen 2009 Cyt. Growth Fact. Rev.20: 475. It is thus preferable for a BMP6 antibody to not bind to BMP2.

BMP2 is further described in, inter alia: Sampath et al. 1990 J. Biol.Chem. 265: 13198; Chen et al. 2004 Growth Factors 22: 233; Marie et al.2002 Histol. Histopath. 17: 877; Nickel et al. 2001 J. Bone Joint Surg.83-A Supp. 1: S7-14; Kirsch et al. 2000 FEBS Lett. 468: 215; Kirsch etal. 2000 EMBO J. 19: 3314; Gilboa et al. 2000 Mol. Biol. Cell 11: 1023.

BMP5 is also a member of the TGF-Beta superfamily. Like other BMPs, itis known for its ability to induce bone and cartilage development. BMP5is expressed in the trabecular meshwork and optic nerve head and mayhave a role in development and normal function. It is also expressed inlung and liver.

Additional information on BMP5 is known in the art, e.g., Hahn et al,1992 Genomics 14: 759; Beck et al. 2003 BMC Neurosci. 2: 12; Celeste etal. 1991 Proc. Natl. Acad. Sci. USA 87: 9843; and Sakaue et al. 1996Biochem. Biophys. Res. Comm. 221: 768.

BMP7 is also a member of the TGF-Beta superfamily. Like other members ofthe BMP family of proteins, it plays a key role in the transformation ofmesenchymal cells into bone and cartilage. It induces thephosphorylation of SMAD1 and SMADS, which in turn induce transcriptionof numerous osteogenic genes.

As noted above, mice with a knock-out of BMP6 are viable and fertile,and show normal bone and cartilage development. However, knock-out micefor BMP7 die after birth with kidney, eye and bone defects. Individualknock-outs of either gene do not alter cardiogenesis, but a doubleknock-out of BMP6 and BMP7 demonstrated several defects and delays inthe heart; embryos died to cardiac insufficiency. BMP7 is important inpreventing progression of chronic heart disease associated withfibrosis, Therefore, cross-reactivity of an anti-BMP6 antibody with BMP7is not desirable.

Additional information related to BMP7 is provided in the art, e.g.,Hahn et al. 1992 Genomics 14: 759; Chen et al. 2004 Growth Factors 22:233; Itoh et al. 2001 EMBO J. 20: 4132; Zeisberg et al. 2003 Am. J.Physiol. Renal Physiol. 285: F1060; Kallui et al. 2009 J. Clin. Invest.119: 1420; and Wang et al. 2001 J. Am. Soc. Neph. 12: 2392.

Hepcidin is a peptide hormone also known as RAMP (Hepcidinanti-microbial protein or peptide).

A recent gene duplication event in mouse evolution has led to thepresence of two similar hepcidin genes in mice, Hepcidin1 and Hepcidin2.Ilyin et al. 2003 FEBS Lett. 542: 22-26. Mouse hepcidin2 lacks severalconserved residues found in mammalian hepcidins. Lou et al. 2004 Blood103: 2816-2821.

The Hepcidin gene product is involved in the maintenance of ironhomeostasis, and it is necessary for the regulation of iron storage inmacrophages, and for intestinal iron absorption. These peptides exhibitantimicrobial activity.

The preproprotein (or preprohormone or preprohepcidin) (84 aa) andproprotein (or prohormone or prohepcidin) (60 aa) are processed intomature peptides of 20, 22 and 25 amino acids. The 25-an peptide issecreted mainly by the liver and is considered the “master regulator” ofiron metabolism. The 20- and 22-aa metabolites exist in the urine. TheN-terminal region of Hepcidin is required for function; deletion of the5 N-terminal amino acids results in loss of function.

The active Hepcidin peptides are rich in cysteines, which formintramolecular bonds that stabilize their beta sheet structures.

Hepcidin is mainly synthesized in the liver, with smaller amounts found.to be synthesized in other tissues. Bekri et al, 2006 Gastroent. 131:788-96.

The 25-aa Hepcidin peptide is secreted mainly by the liver and isconsidered the “master regulator” of iron metabolism. Hepcidin inhibitsiron transport by binding to the iron export channel ferroportin, whichis located on the basolateral surface of gut enterocytes and the plamamembrane of reticuloendothelial cells (macrophages). By inhibitingferroportin, hepcidin prevents enterocytes of the intestines fromsecreting iron ito the hepatic portal system, thereby functionallyreducing iron absorption. The iron release from macrophages is alsoprevented by ferroportin inhibition; therefore, the hepcidin maintainsiron homeostasis. Hepcidin activity is also partially responsible foriron sequestration seen in anemia of chronic inflammation such asinflammatory bowel disease, chronic heart failure, carcinomas,rheumatoid arthritis and renal failure.

Mutations in the hepcidin gene cause hemochromatosis type 2B, also knownas juvenile hemochromatosis, a disease caused by severe iron overloadthat results in cardiomyopathy, cirrhosis, and endocrine failure. Themajority of juvenile hemochromatosis cases are due to mutations inhemojuvelin, a regulator of hepcidin production.

Genetically modified mice engineered to overexpress hepcidin die shortlyafter birth with severe iron deficiency, suggesting a central and notredundant role in iron regulation. The first evidence that linkedhepcidin to anemia of inflammation came when researchers examinedtissues from two patients with liver tumors with a severe microcytic,anemia that did not respond to iron supplements. The tumor tissueoverproduced hepcidin, and removing the tumors surgically cured theanemia,

There are many diseases wherein failure to adequately absorb ironcontributes to iron deficiency and iron deficiency anemia. The treatmentwill depend on the hepcidin levels, as oral treatment will likely beineffective if hepcidin is blocking enteral absorption.

In one embodiment, administration of the antibody or binding fragmentthereof to BMP6 reduces the activity and/or level of Hepcidin and isthus useful in a treatment for anemia. In one embodiment, the inventionpertains to a method of reducing the activity or level of Hepcidin in apatient in need thereof, the method comprising the step of administeringto the patient an antibody or antigen-binding fragment thereof to BMP6.In one embodiment, the activity or level of Hepcidin is reduced by atleast 50%.

Inhibitors to Hepcidin, such as BMP6 antibodies, can be used to treat aHepcidin-related disease. This includes any disease associated withHepcidin and/or a mutation and/or an over-expression of a wild-typeand/or mutant Hepcidin, and/or diseases wherein disease progression isenhanced by or prognosis worsened by the presence of Hepcidin and/or amutation and/or an over-expression of wild-type and/or mutant Hepcidin,and/or reduced renal elimination of hepcidin via the urine. Non-limitingexamples of Hepcidin-related diseases include: anemia, iron-deficienterythropoiesis, hypoferremia, impaired dietary iron uptake, ironsequestration, anemia of inflammation (AI), atherosclerosis, diabetes,and multiple neurodegenerative disorders such as Alzheimer's disease,Parkinson's disease and Friedrich's ataxia, heart failure, chronickidney disease, cardiorenal-anemia syndrome, infection, blood loss,hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism,hemoglobinopathies and malignancies, cancer, AIDS, surgery, stuntedgrowth, and/or hair loss. In one embodiment, the subject is a dialysispatient. In one embodiment, the Hepcidin-related disease is anemia andthe subject is a dialysis patient. The prevalence of iron andESA-refractory anemia is high in chronic hemodialysis population.

Anemia includes, inter alia, anemia of chronic disease (ACD), anemia ofchronic kidney disease (CKD), anemia of cancer, erythropoiesisstimulating agent (ESA) resistant anemia, and/or iron-restricted anemia.

Anemia of CKD is a common and early complication of chronic kidneydisease. Anemia of cancer is caused by hematological malignancies andsome solid tumors. As defined herein, this term also includeschemotherapy-induced anemia, which is anemia caused by chemotherapeuticagents. Anemia in chronic kidney diseases can worsen diabeticneuropathy, cardiovascular disease, retinopathy and other problems.Cancer-related anemia is associated with increased risk of death.

Some chronic diseases such as cancer, kidney disease and autoimmunedisorders can lead to anemia. Overactive inflammatory cytokines cancause dysregulation of iron homeostasis, reduction of erythropoiesis,and a decrease in the life span of red blood cells. Some treatments foranemia include administration of an ESA, erythropoietin, iron (as adietary supplement) or a blood transfusion.

Hepcidin is a key hormone involved in iron homeostasis. High levels ofhepcidin have been associated with iron restricted erythropoiesis inACD. BMP6 is known to increase expression of hepcidin.

Various types of antibodies and antigen-binding fragments thereof toBMP6 are described below.

Homologous Antibodies

In yet another embodiment, the present invention provides an antibody oran antigen-binding fragment thereof comprising amino acid sequences thatare homologous to the sequences described in Table 1 and/or Table 14,and said antibody binds to BMP6, and retains the desired functionalproperties of those antibodies described in Table 1 and/or Table 14.

For example, the invention provides an isolated monoclonal antibody (ora functional antigen-binding fragment thereof) comprising a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises an amino acid sequence that is at least80%, at least 90%, or at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ NOs: 16; 36; 56; or 76; thelight chain variable region comprises an amino acid sequence that is atleast 80%, at least 90%, or at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 26; 46; 66;or 86; the antibody specifically binds to BMP6 protein, and the antibodycan inhibit red blood cell lysis in a hemolytic assay, wherein ahemolytic assay is known in the art. In a specific example, suchantibodies have an IC₅₀ value in a hemolytic assay of 20-200 pM whenusing human BMP6-depleted serum that is reconstituted with 100 pM humanBMP6.

In one embodiment, the VH and/or VL amino acid sequences may be 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequencesset forth in Table 1 and/or Table 14. In one embodiment, the VH and/orVL amino acid sequences may be identical except an amino acidsubstitution in no more than 1, 2, 3, 4 or 5 amino acid position. Anantibody having VH and VL regions having high (i.e., 80% or greater)identity to the VH and VL regions of those described in Table 1 and/orTable 14 can be obtained by mutagenesis (e.g., site-directed orPCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs:16; 36; 56; or 76; and 26; 46; 66; or 86 respectively, followed bytesting of the encoded altered antibody for retained function using thefunctional assays described herein.

In one embodiment, the full length heavy chain and/or full length lightchain amino acid sequences may be 50% 60%, 70%. 80%, 90%, 95%, 96%, 97%,98% or 99% identical to the sequences set forth in Table 1 and/or Table14. An antibody having a full length heavy chain and full length lightchain having high (i.e., 80% or greater) identity to the full lengthheavy chains of any of SEQ ID NOs: 18; 38; 58; or 78 and full lengthlight chains of any of SEQ ID NOs: 28; 48; 68 or 88 respectively, can beobtained by mutagenesis (e.g., site-directed or PCR-mediatedmutagenesis) of nucleic acid molecules encoding such polypeptidesrespectively, followed by testing of the encoded altered antibody forretained function using the functional assays described herein.

In one embodiment, the full length heavy chain and/or full length lightchain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or 99% identical to the sequences set forth in Table 1 and/or Table 14.

In one embodiment, the variable regions of heavy chain and/or lightchain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or 99% identical to the sequences set forth in Table 1 and/or Table 14.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity equals number of identical positions/total number ofpositions X 100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below. Additionallyor alternatively, the protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. For example, suchsearches can be performed using the BLAST program (version 2.0) ofAltschul, et al., 1990 J. Mol. Biol. 215:403-10.

Antibodies with Conservative Modifications

In one embodiment, an antibody of the invention has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the BMP6-binding antibodies and antigen-binding fragmentsthereof of the invention. Accordingly, the invention provides anisolated monoclonal antibody, or a functional antigen-binding fragmentthereof, consisting of a heavy chain variable region comprising CDR1,CDR2, and CDR3 sequences and a light chain variable region comprisingCDR1, CDR2, and CDR3 sequences, wherein: a heavy chain variable regionCDR1 comprising an amino acid sequence selected from any of SEQ ID NOs:29, 49, 69, 12, 32, 52, 72, or 9 or conservative variants thereof; aheavy chain variable region CDR2 comprising an amino acid sequenceselected from any of SEQ ID NOs: 10, 30, 50, 70, 13, 33, 53, or 73 orconservative variants thereof; a heavy chain variable region CDR3comprising an amino acid sequence selected from any of SEQ ID NOs: 11,31, 51, 71, 14, 34, 54, or 74 or conservative variants thereof a lightchain variable region CDRI comprising an amino acid sequence selectedfrom any of SEQ NOs: 19, 39, 59, 79, 22, 42, 62, or 82 or conservativevariants thereof; a light chain variable region CDR2 comprising an aminoacid sequence selected from any of SEQ ID NOs: 20, 40, 60, 80, 23, 43,63, or 83 or conservative variants thereof; and a light chain variableregion CDR3 comprising an amino acid sequence selected from any of SEQID NOs: 21, 41, 61, 81, 24, 44, 64, or 84 or conservative variantsthereof; the antibody or the antigen-binding fragment thereofspecifically binds to BMP6, and inhibits red blood cell lysis in ahemolytic assay.

In one embodiment, an antibody of the invention optimized for expressionin a mammalian cell has a full length heavy chain sequence and a fulllength light chain sequence, wherein one or more of these sequences havespecified amino acid sequences based on the antibodies described hereinor conservative modifications thereof, and wherein the antibodies retainthe desired functional properties of the BMP6-binding antibodies andantigen-binding fragments thereof of the invention. Accordingly, theinvention provides an isolated monoclonal antibody optimized forexpression in a mammalian cell consisting of a full length heavy chainand a full length light chain wherein: the full length heavy chain hasamino acid sequences selected from the group of SEQ ID NOs: 18; 38; 58;or 78, and conservative modifications thereof; and the full length lightchain has amino acid sequences selected from the group of SEQ ID NOs:28; 48; 68 or 88, and conservative modifications thereof the antibodyspecifically binds to BMP6; and the antibody inhibits red blood celllysis in a hemolytic assay as described herein. In a specificembodiment, such antibodies have an IC₅₀ value in a hemolytic assay of20-200 pM when using human BMP6-depleted serum that is reconstitutedwith 100 pM human BMP6.

Antibodies that Bind to the Same Epitope

The present invention provides antibodies that bind to the same epitopeas do the BMP6-binding antibodies listed in Table 1 and/or Table 14. Theepitope bound by Antibody 7 is shown in FIG. 5. Additional antibodiescan therefore be identified based on their ability to cross-compete(e.g., to competitively inhibit the binding of, in a statisticallysignificant manner with other antibodies and antigen-binding fragmentsthereof of the invention in BINTP6 binding assays. The ability of a testantibody to inhibit the binding of antibodies and antigen-bindingfragments thereof of the present invention to BMP6 protein demonstratesthat the test antibody can compete with that antibody for binding toBMP6; such an antibody may, according to non-limiting theory, bind tothe same or a related (e.g., a structurally similar or spatiallyproximal) epitope on the BMP6 as the antibody with which it competes. Ina certain embodiment, the antibody that binds to the same epitope onBMP6 as the antibodies and antigen-binding fragments thereof of thepresent invention is a human monoclonal antibody. Such human monoclonalantibodies can be prepared and isolated as described herein.

Once a desired epitope on an antigen is determined, it is possible togenerate antibodies to that epitope, e.g., using the techniquesdescribed in the present invention. Alternatively, during the discoveryprocess, the generation and characterization of antibodies may elucidateinformation about desirable epitopes. From this information, it is thenpossible to competitively screen antibodies for binding to the sameepitope. An approach to achieve this is to conduct cross-competitionstudies to find antibodies that competitively bind with one another,e.g., the antibodies compete for binding to the antigen. A highthroughput process tier “binning” antibodies based upon theircross-competition is described in International Patent Application No.WO 2003/48731. As will be appreciated by one of skill in the art,practically anything to which an antibody can specifically bind could bean epitope. An epitope can comprises those residues to which theantibody binds. Generally, antibodies specific for a particular targetantigen will preferentially recognize an epitope on the target antigenin a complex mixture of proteins and/or macromolecules.

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. Forexample, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Such techniques are known in the art and described in, e.g.,U.S. Pat. No. 4,708,871; Geysen et al., (1984) Proc. Natl. Acad. Sci.USA 8:3998-4002; Geysen et al., (1985) Proc. Natl. Acad. Sci. USA82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly,conformational epitopes are readily identified by determining spatialconformation of amino acids BMP6such as by, e.g., hydrogen/deuteriumexchange, x-ray crystallography and two-dimensional nuclear magneticresonance. See, e.g., Epitope Mapping Protocols, supra. Antigenicregions of proteins can also be identified using standard antigenicityand hydropathy plots, such as those calculated using, e.g., the Omigaversion 1.0 software program available from the Oxford Molecular Group.This computer program employs the Hopp/Woods method, Hopp et al., (1981)Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicityprofiles, and the Kyte-Doolittle technique, Kyte et al., (1982) J. Mol.Biol. 157:105-132; for hydropathy plots.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the VH and/or VL sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., VH and/or VL), for example within one ormore CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.) Such framework sequences can be obtainedfrom public DNA databases or published references that include germineantibody gene sequences. For example, germine DNA sequences for humanheavy and light chain variable region genes can be found in the “VBase”human germline sequence database (available on the Internet atwww.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al., 1991Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798; and Cox, J. P.L. et al., 1994 Eur. J Immunol. 24:827-836; the contents of each ofwhich are expressly incorporated herein by reference.

An example of framework sequences for use in the antibodies andantigen-binding fragments thereof of the invention are those that arestructurally similar to the framework sequences used by selectedantibodies and antigen-binding fragments thereof of the invention, e.g.,consensus sequences and/or framework sequences used by monoclonalantibodies of the invention. The VH CDR1, 2 and 3 sequences, and the VLCDR1, 2 and 3 sequences, can be grafted onto framework regions that havethe identical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Grafting Antigen-Binding Domains Into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to BMP6. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins,antigen-binding fragments thereof, and include immunoglobulins of otheranimal species, preferably having humanized aspects. Single heavy-chainantibodies such as those identified in camelids are of particularinterest in this regard. Novel frameworks, scaffolds and fragmentscontinue to be discovered and developed by those skilled in the art.

In one aspect, the invention pertains to a method of generatingnon-imnumoglobulin based antibodies using non-immunoglobulin scaffoldsonto which CDRs of the invention can be grafted. Known or futurenon-immunoglobulin frameworks and scaffolds may be employed, as long asthey comprise a binding region specific for the target BMP6 protein.Known non-immunoglobulin frameworks or scaffolds include, but are notlimited to, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.),ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (SciI. ProteinsGmbH, Halle, Germany).

The fibronectin scaffolds are based on fibronectin type III domain(e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).The fibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). These fibronectin-based scaffolds are not an immunoglobulin,although the overall fold is closely related to that of the smallestfunctional antibody fragment, the variable region of the heavy chain,which comprises the entire antigen recognition unit in camel and llamaIgG. Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity forthose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat module is a 33amino acid polypeptide consisting of two anti-parallel alpha-helices anda beta-turn. Binding of the variable regions is mostly optimized byusing ribosome display.

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, U.S.Patent Application Publication Nos. 20040175756; 20050053973;20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids, The proteinarchitecture is reminiscent of immunoglobulins, with hypervariable loopson top of a rigid framework. However, in contrast with antibodies ortheir recombinant fragments, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues, being justmarginally bigger than a single immunoglobulin domain. The set of fourloops, which makes up the binding pocket, shows pronounced structuralplasticity and tolerates a variety of side chains. The binding site canthus be reshaped in a proprietary process in order to recognizeprescribed target molecules of different shape with high affinity andspecificity. One protein of lipocalin family, the bilin-binding protein(BBP) of Pieris Brassicae has been used to develop anticalins bymutagenizing the set of four loops. One example of a patent applicationdescribing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New affilin molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin molecules do not show any structural homology to immunoglobulinproteins. Currently, two affilin scaffolds are employed, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368. Protein epitopemimetics (PEM) are medium-sized, cyclic, peptide-like molecules (MW 1-2kDa) mimicking beta-hairpin secondary structures of proteins, the majorsecondary structure involved in protein-protein interactions.

The human BMP6-binding antibodies can be generated using methods thatare known in the art. For example, the humaneering technology used toconverting non-human antibodies into engineered human antibodies. U.S.Patent Publication No. 20050008625 describes an in vivo method forreplacing a nonhuman antibody variable region with a human variableregion in an antibody while maintaining the same or providing betterbinding characteristics relative to that of the nonhuman antibody. Themethod relies on epitope guided replacement of variable regions of anon-human reference antibody with a fully human antibody. The resultinghuman antibody is generally unrelated structurally to the referencenonhuman antibody, but binds to the same epitope on the same antigen asthe reference antibody. Briefly, the serial epitope-guidedcomplementarity replacement approach is enabled by setting up acompetition in cells between a “competitor” and a library of diversehybrids of the reference antibody (“lest antibodies”) for binding tolimiting amounts of antigen in the presence of a reporter system whichresponds to the binding of test antibody to antigen. The competitor canbe the reference antibody or derivative thereof such as a single-chainFv fragment. The competitor can also be a natural or artificial ligandof the antigen which binds to the same epitope as the referenceantibody. The only requirements of the competitor are that it binds tothe same epitope as the reference antibody, and that it competes withthe reference antibody for antigen binding. The test antibodies have oneantigen-binding V-region in common from the nonhuman reference antibody,and the other V-region selected at random from a diverse source such asa repertoire library of human antibodies. The common V-region from thereference antibody serves as a guide, positioning the test antibodies onthe same epitope on the antigen, and in the same orientation, so thatselection is biased toward the highest antigen-binding fidelity to thereference antibody.

Many types of reporter system can be used to detect desired interactionsbetween test antibodies and antigen. For example, complementing reporterfragments may be linked to antigen and test antibody, respectively, sothat reporter activation by fragment complementation only occurs whenthe test antibody binds to the antigen. When the test antibody- andantigen-reporter fragment fusions are co-expressed with a competitor,reporter activation becomes dependent on the ability of the testantibody to compete with the competitor, which is proportional to theaffinity of the test antibody for the antigen. Other reporter systemsthat can be used include the reactivator of an auto-inhibited reporterreactivation system (RAIR) as disclosed in U.S. patent application Ser.No. 10/208,730 (Publication No. 20030198971), or competitive activationsystem disclosed in U.S. patent application Ser. No. 10/076,845(Publication No, 20030157579).

With the serial epitope-guided complementarity replacement system,selection is made to identify cells expresses a single test antibodyalong with the competitor, antigen, and reporter components. In thesecells, each test antibody competes one-on-one with the competitor forbinding to a limiting amount of antigen. Activity of the reporter isproportional to the amount of antigen bound to the test antibody, whichin turn is proportional to the affinity of the test antibody for theantigen and the stability of the test antibody. Test antibodies areinitially selected on the basis of their activity relative to that ofthe reference antibody when expressed as the test antibody. The resultof the first round of selection is a set of “hybrid” antibodies, each ofwhich is comprised of the same non-human V-region from the referenceantibody and a human V-region from the library, and each of which bindsto the same epitope on the antigen as the reference antibody. One ofmore of the hybrid antibodies selected in the first round will have anaffinity for the antigen comparable to or higher than that of thereference antibody.

In the second V-region replacement step, the human V-regions selected inthe first step are used as guide for the selection of human replacementsfor the remaining non-human reference antibody V-region with a diverselibrary of cognate human V-regions. The hybrid antibodies selected inthe first round may also be used as competitors for the second round ofselection. The result of the second round of selection is a set of fullyhuman antibodies which differ structurally from the reference antibody,but which compete with the reference antibody for binding to the sameantigen. Some of the selected human antibodies bind to the same epitopeon the same antigen as the reference antibody. Among these selectedhuman antibodies, one or more binds to the same epitope with an affinitywhich is comparable to or higher than that of the reference antibody.

Using one of the mouse or chimeric BMP6-binding antibodies describedabove as the reference antibody, this method can be readily employed togenerate human antibodies that bind to human BMP6 with the same bindingspecificity and the same or better binding affinity. In addition, suchhuman BMP6-binding antibodies can also be commercially obtained fromcompanies which customarily produce human antibodies, e.g., KaloBios,Inc. (Mountain View, Calif.).

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschherger, M. et al. 2003 Bioconjugate Chem. 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries ofcamelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies andantigen-binding fragments thereof of non-human origin, an amino acidsequence of a camelid antibody can be altered recombinantly to obtain asequence that more closely resembles a human sequence, i.e., thenanobody can be “humanized”. Thus the natural low antigenicity ofcamelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for BMP6. In one embodiment herein, thecamelid antibody or nanobody is naturally produced in the camelidanimal, is produced by the camelid following immunization with BMP6 or apeptide fragment thereof, using techniques described herein for otherantibodies. Alternatively, the BMP6-binding camelid nanobody isengineered, i.e., produced by selection for example from a library ofphage displaying appropriately mutagenized camelid nanobody proteinsusing panning procedures with BMP6 as a target as described in theexamples herein. Engineered nanobodies can further be customized bygenetic engineering to have a half life in a recipient subject of from45 minutes to two weeks. In a specific embodiment, the camelid antibodyor nanobody is obtained by grafting the CDRs sequences of the heavy orlight chain of the human antibodies of the invention into nanobody orsingle domain antibody framework sequences, as described for example inPCT/EP93/02214.

Bispecific Molecules and Multivalent Antibodies

In another aspect, the present invention features bispecific ormultispecific molecules comprising an BMP6-binding antibody, or afragment thereof, of the invention. An antibody of the invention, orantigen-binding regions thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the invention may in fact be derivatized or linked tomore than one other functional molecule to generate multi-specificmolecules that bind to more than two different binding sites and/ortarget molecules; such multi-specific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule of the invention, an antibody of the invention canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for BMP6 and a secondbinding specificity for a second target epitope. For example, the secondtarget epitope is another epitope of BMP6 different from the firsttarget epitope.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778.

Diabodies are bivalent, bispecific molecules in which VH and VL domainsare expressed on a single polypeptide chain, connected by a linker thatis too short to allow for pairing between the two domains on the samechain. The VH and VL domains pair with complementary domains of anotherchain, thereby creating two antigen binding sites (see e.g., Holliger etal., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poijak et al., 1994Structure 2:1121-1123). Diabodies can be produced by expressing twopolypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VLconfiguration), or VLA-VHB and VLB-VHA (VL-VH configuration) within thesame cell. Most of them can be expressed in soluble form in bacteria.Single chain diabodies (scDb) are produced by connecting the twodiabody-forming polypeptide chains with linker of approximately 15 aminoacid residues (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45 (3-4):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36). scDb can be expressed in bacteria in soluble, activemonomeric form (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45 (34): 128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3 (2): 83-105;Ridgway et al., 1996 Protein Eng., 9 (7):617-21). A diabody can be fusedto Fc to generate a “di-diabody” (see Lu et al., 2004 J. Biol. Chem.,279 (4):2856-65).

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb X mAb, mAb XFab, Fab X F(ab′)2 or ligand X Fab fusion protein. A bispecific moleculeof the invention can be a single chain molecule comprising one singlechain antibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

In another aspect, the present invention provides multivalent compoundscomprising at least two identical or different antigen-binding portionsof the antibodies and antigen-binding fragments thereof of the inventionbinding to BMP6. The antigen-binding portions can be linked together viaprotein fusion or covalent or non covalent linkage. Alternatively,methods of linkage has been described for the bispecific molecules.Tetravalent compounds can be obtained for example by cross-linkingantibodies and antigen-binding fragments thereof of the invention withan antibody or antigen-binding fragment that binds to the constantregions of the antibodies and antigen-binding fragments thereof of theinvention, for example the Fc or hinge region.

Trimerizing domain are described for example in Borean patent EP 1 012280B1. Pentamerizing modules are described for example inPCT/EP97/05897.

Antibodies with Extended Half Life

The present invention provides for antibodies that specifically bind toBMP6 which have an extended half-life in vivo.

Many factors may affect a protein's half life in vivo. For examples,kidney filtration, metabolism in the liver, degradation by proteolyticenzymes (proteases), and immunogenic responses (e.g., proteinneutralization by antibodies and uptake by macrophages and dentriticcells). A variety of strategies can be used to extend the half life ofthe antibodies and antigen-binding fragments thereof of the presentinvention. For example, by chemical linkage to polyethyleneglycol (PEG),reCODE PEG, antibody scaffold, polysialic acid (PSA), hydroxyethylstarch (HES), albumin-binding ligands, and carbohydrate shields; bygenetic fusion to proteins binding to serum proteins, such as albumin,IgG, FcRn, and transferring; by coupling (genetically or chemically) toother binding moieties that bind to serum proteins, such as nanobodies,Fabs, DARPins, avimers, affibodies, and anticalins; by genetic fusion torPEG, albumin, domain of albumin, albumin-binding proteins, and Fc; orby incorporation into nancarriers, slow release formulations, or medicaldevices.

To prolong the serum circulation of antibodies in vivo, inert polymermolecules such as high molecular weight PEG can be attached to theantibodies or a fragment thereof with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of the antibodies or via epsilon-amino groups present onlysine residues. To pegylate an antibody, the antibody, antigen-bindingfragment thereof, typically is reacted with polyethylene glycol (PEG),such as a reactive ester or aldehyde derivative of PEG, under conditionsin which one or more PEG groups become attached to the antibody orantibody fragment. The pegylation can be carried out by an acylationreaction or an alkylation reaction with a reactive PEG molecule (or ananalogous reactive water-soluble polymer). As used herein, the term“polyethylene glycol” is intended to encompass any of the forms of PEGthat have been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In one embodiment, the antibody to be pegylated is anaglycosylated antibody. Linear or branched polymer derivatization thatresults in minimal loss of biological activity will be used. The degreeof conjugation can be closely monitored by SDS-PAGE and massspectrometry to ensure proper conjugation of PEG molecules to theantibodies. Unreacted PEG can be separated from antibody-PEG conjugatesby size-exclusion or by ion-exchange chromatography. PEG-derivatizedantibodies can be tested for binding activity as well as for in vivoefficacy using methods well-known to those of skill in the art, forexample, by immunoassays described herein. Methods for pegylatingproteins are known in the art and can be applied to the antibodies andantigen-binding fragments thereof of the invention. See for example, EP0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other modified pegylation technologies include reconstituting chemicallyorthogonal directed engineering technology (ReCODE PEG), whichincorporates chemically specified side chains into biosynthetic proteinsvia a reconstituted system that includes tRNA synthetase and tRNA. Thistechnology enables incorporation of more than 30 new amino acids intobiosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNAincorporates a normative amino acid any place an amber codon ispositioned, converting the amber from a stop codon to one that signalsincorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serumhalflife extension. This technology involves genetically fusing a300-600 amino acid unstructured protein tail to an existingpharmaceutical protein. Because the apparent molecular weight of such anunstructured protein chain is about 15-fold larger than its actualmolecular weight, the serum halflife of the protein is greatlyincreased. In contrast to traditional PEGylation, which requireschemical conjugation and repurification, the manufacturing process isgreatly simplified and the product is homogeneous.

Polysialytion is another technology, which uses the natural polymerpolysialic acid (PSA) to prolong the active life and improve thestability of therapeutic peptides and proteins. PSA is a polymer ofsialic acid (a sugar). When used for protein and therapeutic peptidedrug delivery, polysialic acid provides a protective microenvironment onconjugation. This increases the active life of the therapeutic proteinin the circulation and prevents it from being recognized by the immunesystem. The PSA polymer is naturally found in the human body. It wasadopted by certain bacteria which evolved over millions of years to coattheir walls with it. These naturally polysialylated bacteria were thenable, by virtue of molecular mimicry, to foil the body's defense system.PSA, nature's ultimate stealth technology, can be easily produced fromsuch bacteria in large quantities and with predetermined physicalcharacteristics. Bacterial PSA is completely non-immunogenic, even whencoupled to proteins, as it is chemically identical to PSA in the humanbody.

Another technology include the use of hydroxyethyl starch (“HES”)derivatives linked to antibodies. HES is a modified natural polymerderived from waxy maize starch and can be metabolized by the body'senzymes. HES solutions are usually administered to substitute deficientblood volume and to improve the rheological properties of the blood.Hesylation of an antibody enables the prolongation of the circulationhalf-life by increasing the stability of the molecule, as well as byreducing renal clearance, resulting in an increased biological activity.By varying different parameters, such as the molecular weight of HES, awide ranee of HES antibody conjugates can be customized.

Antibodies having an increased half-life in vivo can also be generatedintroducing one or more amino acid modifications (i.e., substitutions,insertions or deletions) into an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge Fc domain fragment). See,e.g., International Publication No. WO 98/23289; InternationalPublication No. WO 97/34631; and U.S. Pat. No. 6,277,375. Further,antibodies can be conjugated to albumin in order to make the antibody orantibody fragment more stable in vivo or have a longer half life invivo. The techniques are well-known in the art, see, e.g., InternationalPublication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and EuropeanPatent No. EP 413,622.

The strategies for increasing half life is especially useful innanobodies, fibronectin-based binders, and other antibodies or proteinsfor which increased in vivo half life is desired.

Antibody Conjugates

The present invention provides antibodies or antigen-binding fragmentsthereof that specifically bind to BMP6 recombinantly fused or chemicallyconjugated. (including both covalent and non-covalent conjugations) to aheterologous protein or polypeptide (or antigen-binding fragmentthereof, preferably to a polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids) to generate fusionproteins. In particular, the invention provides fusion proteinscomprising an antigen-binding fragment of an antibody described herein(e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, a VHdomain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein,polypeptide, or peptide. Methods for fusing or conjugating proteins,polypeptides, or peptides to an antibody or an antibody fragment areknown in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929,5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP307,434 and EP 367,166; International Publication Nos. WO 96/04388 andWO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil etal., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies and antigen-bindingfragments thereof of the invention (e.g., antibodies and antigen-bindingfragments thereof with higher affinities and lower dissociation rates).See, generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol. 16 (2):76-82; Hansson, etal., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998,Biotechniques 24 (2):308-313 (each of these patents and publications arehereby incorporated by reference in its entirety). Antibodies andantigen-binding fragments thereof, or the encoded antibodies andantigen-binding fragments thereof, may be altered by being subjected torandom mutagenesis by error-prone PCR, random nucleotide insertion orother methods prior to recombination. A polynucleotide encoding anantibody antigen-binding fragment thereof that specifically binds toBMP6 may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies and antigen-binding fragments thereof can befused to marker sequences, such as a peptide to facilitate purification.In one embodiment, the marker amino acid sequence is a hexa-histidinepeptide (SEQ ID NO: 97), such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance,hexa-histidine (SEQ ID NO: 97) provides for convenient purification ofthe fusion protein. Other peptide tags useful for purification include,but are not limited to, the hemagglutinin (“HA”) tag, which correspondsto an epitope derived from the influenza hemagglutinin protein (Wilsonet al., 1984, Cell 37:767), and the “flag” tag.

In one embodiment, antibodies and antigen-binding fragments thereof ofthe present invention antigen-binding fragments thereof conjugated to adiagnostic or detectable agent. Such antibodies can be useful formonitoring or prognosing the onset, development, progression and/orseverity of a disease or disorder as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.Such diagnosis and detection can accomplished by coupling the antibodyto detectable substances including, but not limited to, various enzymes,such as, but not limited to, horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as, but not limited to, streptavidin/biotin andavidin/biotin; fluorescent materials, such as, but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as, but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as, but not limitedto, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S),tritium (3H), indium (115In, 113In, 112In, and 111In), technetium(99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd),molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd,149 Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh,97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se,113Sn, and 117Tin; and positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions.

The present invention further encompasses uses of antibodies andantigen-binding fragments thereof conjugated to a therapeutic moiety. Anantibody antigen-binding fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters. A cytotoxin or cytotoxic agent includes any agent thatis detrimental to cells.

Further, an antibody antigen-binding fragment thereof may be conjugatedto a therapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor,alpha-interferon, beta-interferon, nerve growth factor, platelet derivedgrowth factor, tissue plasminogen activator, an apoptotic agent, ananti-angiogenic agent; or, a biological response modifier such as, forexample, a lymphokine.

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, such as alpha-emitters such as 213Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, 131In, 131LU, 131Y, 131Ho, 131Sm, to polypeptides.In one embodiment, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4 (10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10 (4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Antibodies may also be attached to solid supports, which areparticularly useful for .immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Methods of Producing Antibodies of the Invention Nucleic Acids Encodingthe Antibodies

The invention provides substantially purified nucleic acid moleculeswhich encode polypeptides comprising segments or domains of theBMP6-binding antibody chains described above. Some of the nucleic acidsof the invention comprise the nucleotide sequence encoding the heavychain variable region shown in any of SEQ NOs: 16; 36; 56; or 76, and/orthe nucleotide sequence encoding the light chain variable region shownin any of SEQ ID NOs: 26; 46; 66; or 86. in a specific embodiment, thenucleic acid molecules are those identified in Table 1. Some othernucleic acid molecules of the invention comprise nucleotide sequencesthat are substantially identical (e.g., at least 65, 80%, 95%, or 99%)to the nucleotide sequences of those identified in Table 1. Whenexpressed from appropriate expression vectors, polypeptides encoded bythese polynucleotides are capable of exhibiting BMP6 antigen bindingcapacity.

Also provided in the invention are polynucleotides which encode at leastone CDR region and usually all three CDR regions from the heavy or lightchain of the BMP6-binding antibody set forth in Table 1 and/or Table 14.Some other polynucleotides encode all or substantially all of thevariable region sequence of the heavy chain and/or the light chain ofthe BMP6-binding antibody set forth in Table 1 and/or Table 14. Becauseof the degeneracy of the code, a variety of nucleic acid sequences willencode each of the immunoglobulin amino acid sequences.

The nucleic acid molecules of the invention can encode both a variableregion and a constant region of the antibody. Some of nucleic acidsequences of the invention comprise nucleotides encoding a mature heavychain variable region sequence that is substantially identical (e.g., atleast 80%, 90%, or 99%) to the mature heavy chain variable regionsequence set forth in any of SEQ ID NOs: 16; 36; 56; or 76. Some othernucleic acid sequences comprising nucleotide encoding a mature lightchain variable region sequence that is substantially identical at least80%, 90%, or 99%) to the mature light chain variable region sequence setforth in any of SEQ ID NOs: 26; 46; 66; or 86.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding an BMP6-binding antibody orits binding fragment. Direct chemical synthesis of nucleic acids can beaccomplished by methods known in the art, such as the phosphodiestermethod of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g, PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications,Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila etal., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the invention are expression vectors and host cells forproducing the BMP6-binding antibodies described above. Variousexpression vectors can be employed to express the polynucleotidesencoding the BMP6-binding antibody chains or binding fragments. Bothviral-based and nonviral expression vectors can be used to produce theantibodies in a mammalian host cell. Nonviral vectors and systemsinclude plasmids, episomal vectors, typically with an expressioncassette for expressing a protein or RNA, and human artificialchromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997). Forexample, nonviral vectors useful for expression of the BMP6-bindingpolynucleotides and polypeptides in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors includevectors based on retroviruses, adenoviruses, adenoassociated viruses,herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barrvirus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brentet al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeldet al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding an BMP6-bindingantibody chain antigen-binding fragment. In one embodiment, an induciblepromoter is employed to prevent expression of inserted sequences exceptunder inducing conditions. Inducible promoters include, e.g., arabinose,lacZ, metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of an BMP6-binding antibody chain antigen-bindingfragment. These elements typically include an ATG initiation codon andadjacent ribosome binding site or other sequences. In addition, theefficiency of expression may be enhanced by the inclusion of enhancersappropriate to the cell system in use see, e.g., Scharf et al., ResultsProbl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol.,153:516, 1987). For example, the SV40 enhancer or CMV enhancer may beused to increase expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedBMP6-binding antibody sequences. More often, the inserted BMP6-bindingantibody sequences are linked to a signal sequences before inclusion inthe vector. Vectors to be used to receive sequences encodingBMP6-binding antibody light and heavy chain variable domains sometimesalso encode constant regions or parts thereof. Such vectors allowexpression of the variable regions as fusion proteins with the constantregions thereby leading to production of intact antibodies andantigen-binding fragments thereof. Typically, such constant regions arehuman.

The host cells for harboring and expressing the BMP6-binding antibodychains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters typicallycontrol expression, optionally with an operator sequence, and haveribosome binding site sequences and the like, for initiating andcompleting transcription and translation. Other microbes, such as yeast,can also be employed to express BMP6-binding polypeptides of theinvention. Insect cells in combination with baculovirus vectors can alsobe used.

In one embodiment, mammalian host cells are used to express and producethe BMP6-binding polypeptides of the present invention. For example,they can be either a hybridoma cell line expressing endogenousimmunoglobulin genes (e.g., the 1D6.C9 myeloma hybridoma clone asdescribed in the Examples) or a mammalian cell line harboring anexogenous expression vector (e.g., the SP2/0 myeloma cells exemplifiedbelow). These include any normal mortal or normal or abnormal immortalanimal or human cell. For example, a number of suitable host cell linescapable of secreting intact immunoglobulins have been developedincluding the CHO cell lines, various Cos cell lines, HeLa cells,myeloma cell lines, transformed B-cells and hybridomas. The use ofmammalian tissue cell culture to express polypeptides is discussedgenerally in, e.g., Winnacker, FROM GENES TO CLONES, VCH Publishers,N.Y., N.Y., 1987. Expression vectors for mammalian host cells caninclude expression control sequences, such as an origin of replication,a promoter, and an enhancer (see, e.g., Queen, et al., Immunol. Rev.89:49-68, 1986), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. These expression vectors usuallycontain promoters derived from mammalian genes or from mammalianviruses. Suitable promoters may be constitutive, cell type-specific,stage-specific, and/or modulatable or regulatable. Useful promotersinclude, but are not limited to, the metallothionein promoter, theconstitutive adenovirus major late promoter, the dexamethasone-inducibleMMTV promoter, the SV40 promoter, the MRP poIIII promoter, theconstitutive MPSV promoter, the tetracycline-inducible CMV promoter(such as the human immediate-early CMV promoter), the constitutive CMVpromoter, and promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts. (See generallySambrook, et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express BMP6-binding antibody chains or bindingfragments can be prepared using expression vectors of the inventionwhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype.

Generation of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known. Chimeric orhumanized antibodies and antigen-binding fragments thereof of thepresent invention can be prepared based on the sequence of a murinemonoclonal antibody prepared as described above. DNA encoding the heavyand light chain immunoglobulins can be obtained from the murinehybridoma of interest and engineered to contain non-murine (e.g., human)immunoglobulin sequences using standard molecular biology techniques.For example, to create a chimeric antibody, the murine variable regionscan be linked to human constant regions using methods known in the art(see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create ahumanized antibody, the murine CDR regions can be inserted into a humanframework using methods known in the art. See e.g., U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762and 6,180,370 to Queen et al.

In a certain embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstBMP6 can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHuMAb mice and KM mice, respectively, and are collectively referred toherein as “human Ig mice.” The HuMAb Mouse® (Medarex, Inc.) containshuman immunoglobulin gene miniloci that encode un-rearranged human heavy(mu and gamma) and kappa light chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous mu and kappachain loci (see e.g., Lonberg, et al., 1994 Nature 368 (6474): 856-859).Accordingly, the mice exhibit reduced expression of mouse IgM or K, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgG-kappa monoclonal (Lonberg, N. et al., 1994 supra;reviewed in Lonberg, N., 1994 Handbook of Experimental Pharmacology113:49-101; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N., 1995 Ann. N.Y. Acad. Sci.764:536-546). The preparation and use of HuMAb mice, and the genomicmodifications carried by such mice, is further described in Taylor, L.et al., 1992 Nucleic Acids Research 20:6287-6295: Chen. J. et al., 1993International Immunology 5: 647-656; Tuaillon et al., 1993 Proc. Natl.Acad. Sci. USA 94:3720-3724; Choi et al., 1993 Nature Genetics4:117-123; Chen, J. et al., 1993 EMBO J. 12: 821-830; Tuaillon et al.,1994 J. Immunol. 152:2912-2920; Taylor, L. et al., 1994 InternationalImmunology 579-591; and Fishwild, D. et al., 1996 Nature Biotechnology14: 845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92103918, WO 93/12227, WO 94/25585, WO 97113852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseBMP6-binding antibodies and antigen-binding fragments thereof of theinvention. For example, an alternative transgenic system referred to asthe Xenomouse (Abgenix, Inc.) can be used. Such mice are described in,e.g., U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseBMP6-binding antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raise BMP6-bindingantibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art or described in the examples below. See forexample: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner etal; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al; U.S. Pat.Nos. 5,969,108 and 6,172,197 to McCafferty et al; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Framework or Fc Engineering

Engineered antibodies and antigen-binding fragments thereof of theinvention include those in which modifications have been made toframework residues within VH and/or VL, e.g. to improve the propertiesof the antibody. Typically such framework modifications are made todecrease the immunogenicity of the antibody. For example, one approachis to “backmutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived. To return theframework region sequences to their germline configuration, the somaticmutations can be “backmutated” to the germline sequence by, for example,site-directed mutagenesis. Such “backmutated” antibodies are alsointended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al. In addition or alternative tomodifications made within the framework or CDR regions, antibodies ofthe invention may be engineered to include modifications within the Fcregion, typically to alter one or more functional properties of theantibody, such as serum half-life, complement fixation, Fc receptorbinding, and/or antigen-dependent cellular cytotoxicity. Furthermore, anantibody of the invention may be chemically modified (e.g., one or morechemical moieties can be attached to the antibody) or be modified toalter its glycosylation, again to alter one or more functionalproperties of the antibody. Each of these embodiments is described infurther detail below. The numbering of residues in the Fc region is thatof the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In one embodiment, the Fc region is altered by replacing at least oneamino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fc-gamma receptor by modifying one or more amino acids. This approachis described further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgGI for Fc-gamma RI, Fc-gamma RII, Fc-gammaRIII and FcRn have been mapped and variants with improved binding havebeen described (see Shields, R. L. et al., 2001 J. Biol. Chen.276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen”. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, LecI3 cells, with reducedability to attach fucose to Asn (297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta (1,4)-N acetylglucosaminyltransferase III (GnTIII)) suchthat antibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Methods of Engineering Altered Antibodies

As discussed above, the BMP6-binding antibodies having VH and VLsequences or full length heavy and light chain sequences shown hereincan be used to create new BMP6-binding antibodies by modifying fulllength heavy chain and/or light chain sequences, VH and/or VL sequences,or the constant region(s) attached thereto. Thus, in another aspect ofthe invention, the structural features of BMP6-binding antibody of theinvention are used to create structurally related BMP6-bindingantibodies that retain at least one functional property of theantibodies and antigen-binding fragments thereof of the invention, suchas binding to human BMP6 and also inhibiting one or more functionalproperties of BMP6 (e.g., inhibit red blood cell lysis in a hemolyticassay).

For example, one or more CDR regions of the antibodies andantigen-binding fragments thereof of the present invention, or mutationsthereof, can be combined recombinantly with known framework regionsand/or other CDRs to create additional, recombinantly-engineered,BMP6-binding antibodies and antigen-binding fragments thereof of theinvention, as discussed above. Other types of modifications includethose described in the previous section. The starting material for theengineering method is one or more of the VH and/or VL sequences providedherein, or one or more CDR regions thereof. To create the engineeredantibody, it is not necessary to actually prepare (i.e., express as aprotein) an antibody having one or more of the VH and/or VL sequencesprovided herein, or one or more CDR regions thereof. Rather, theinformation contained in the sequence(s) is used as the startingmaterial to create a “second generation” sequence(s) derived from theoriginal sequence(s) and then the “second generation” sequence(s) isprepared and expressed as a protein. The altered antibody sequence canalso be prepared by screening antibody libraries having fixed CDR3sequences or minimal essential binding determinants as described inUS20050255552 and diversity on CDR1 and CDR2 sequences. The screeningcan be performed according to any screening technology appropriate forscreening antibodies from antibody libraries, such as phage displaytechnology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the BMP6-binding antibodies described herein,which functional properties include, but are not limited to,specifically binding to human BMP6 protein; and the antibody inhibit redblood cell lysis in a hemolytic assay.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

In one embodiment of the methods of engineering antibodies andantigen-binding fragments thereof of the invention, mutations can beintroduced randomly or selectively along all or part of an BMP6-bindingantibody coding sequence and the resulting modified BMP6-bindingantibodies can be screened for binding activity and/or other functionalproperties as described herein. Mutational methods have been describedin the art. For example, PCT Publication WO 02/092780 by Short describesmethods for creating and screening antibody mutations using saturationmutagenesis, synthetic ligation assembly, or a combination thereof.Alternatively, PCT Publication WO 03/074679 by Lazar et al. describesmethods of using computational screening methods to optimizephysiochemical properties of antibodies.

Characterization of the Antibodies of the Invention

The antibodies and antigen-binding fragments thereof of the inventioncan be characterized by various functional assays. For example, they canbe characterized by their ability to inhibit BMP6.

The ability of an antibody to bind to BMP6 can be detected by labellingthe antibody of interest directly, or the antibody may be unlabelled andbinding detected indirectly using various sandwich assay formats knownin the art.

In one embodiment, the BMP6-binding antibodies and antigen-bindingfragments thereof of the invention block or compete with binding of areference BMP6-binding antibody to BMP6 polypeptide. These can be fullyhuman BMP6-binding antibodies described above. They can also be othermouse, chimeric or humanized BMP6-binding antibodies which bind to thesame epitope as the reference antibody. The capacity to block or competewith the reference antibody binding indicates that BMP6-binding antibodywider test binds to the same or similar epitope as that defined by thereference antibody, or to an epitope which is sufficiently proximal tothe epitope bound by the reference BMP6-binding antibody. Suchantibodies are especially likely to share the advantageous propertiesidentified for the reference antibody. The capacity to block or competewith the reference antibody may he determined by, e.g., a competitionbinding assay. With a competition binding assay, the antibody under testis examined for ability to inhibit specific binding of the referenceantibody to a common antigen, such as BMP6 polypeptide. A test antibodycompetes with the reference antibody for specific binding to the antigenif an excess of the test antibody substantially inhibits binding of thereference antibody. Substantial inhibition means that the test antibodyreduces specific binding of the reference antibody usually by at least10%, 25%, 50%, 75%, or 90%.

There are a number of known competition binding assays that can be usedto assess competition of an antibody with a reference antibody forbinding to a particular protein, in this case, BMP6. These include,e.g., solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA), sandwich competition assay(see Stahli et al., Methods in Enzymology 9:242-253, 1983); solid phasedirect biotin-avidin EIA (see Kirkland et al., J. Immunol.137:3614-3619, 1986); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see Harlow & Lane, supra); solid phasedirect label RIA using I-125 label (see Morel et al., Molec. Immunol.25:7-15, 1988); solid phase direct biotin-avidin EIA (Cheung et al.,Virology 176:546-552, 1990); and direct labeled RIA (Moldenhauer et al.,Scand. J. Immunol. 32:77-82, 1990). Typically, such an assay involvesthe use of purified antigen bound to a solid surface or cells bearingeither of these, an unlabelled test BMP6-binding antibody and a labelledreference antibody. Competitive inhibition is measured by determiningthe amount of label bound to the solid surface or cells in the presenceof the test antibody. Usually the test antibody is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur.

To determine if the selected BMP6-binding monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (e.g., reagents from Pierce, Rockford, Ill.).Competition studies using unlabeled monoclonal antibodies andbiotinylated monoclonal antibodies can he performed using BMP6polypeptide coated-ELISA plates. Biotinylated MAb binding can bedetected with a strep-avidin-alkaline phosphatase probe. To determinethe isotype of a purified BMP6-binding antibody, isotype ELISAs can beperformed. For example, wells of microtiter plates can be coated with 1□g/ml of anti-human IgG overnight at 4 degrees C. After blocking with 1%BSA, the plates are reacted with 1 □g/ml or less of the monoclonalBMP6-binding antibody or purified isotype controls, at ambienttemperature for one to two hours. The wells can then be reacted witheither human IgG1 or human IgM-specific alkaline phosphatase-conjugatedprobes. Plates are then developed and analyzed so that the isotype ofthe purified antibody can be determined.

To demonstrate binding of monoclonal BMP6-binding antibodies to livecells expressing BMP6 polypeptide, flow cytometry can be used. Briefly,cell lines expressing BMP6 (grown under standard growth conditions) canbe mixed with various concentrations of BMP6-binding antibody in PBScontaining 0.1% BSA and 10% fetal calf serum, and incubated at 37degrees C. for 1 hour. After washing, the cells are reacted withFluorescein-labeled anti-human IgG antibody under the same conditions asthe primary antibody staining. The samples can be analyzed by FACScaninstrument using light and side scatter properties to gate on singlecells. An alternative assay using fluorescence microscopy may be used(in addition to or instead of) the flow cytometry assay. Cells can bestained exactly as described above and examined by fluorescencemicroscopy. This method allows visualization of individual cells, butmay have diminished sensitivity depending on the density of the antigen.BMP6-binding antibodies and antigen-binding fragments thereof of theinvention can be further tested for reactivity with BMP6 polypeptide orantigenic fragment by Western blotting. Briefly, purified BMP6polypeptides or fusion proteins, or cell extracts from cells expressingBMP6 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens are transferred to nitrocellulose membranes, blocked with 10%fetal calf serum, and probed with the monoclonal antibodies to betested. Human IgG binding can be detected using anti-human IgG alkalinephosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem.Co., St. Louis, Mo.). Examples of functional assays are also describedin the Example section below.

Prophylactic and Therapeutic Uses

The present invention provides methods of treating a disease or disorderassociated with increased BMP6 activity by administering to a subject inneed thereof an effective amount of the antibodies and antigen-bindingfragments thereof of the invention. In a specific embodiment, thepresent invention provides a method of treating anemia by administeringto a subject in need thereof an effective amount of the antibodies andantigen-binding fragments thereof of the invention. The antibodies andantigen-binding fragments thereof of the invention can be used, interalia to prevent progression of anemia. It can also be used incombination with other therapies for the treatment of anemia patients.

In one embodiment, the present invention provides methods of treating aBMP6 related disease or disorder by administering to a subject in needthereof an effective amount of the antibodies and antigen-bindingfragments thereof of the invention. Examples of known BMP6 relateddiseases or disorders include: anemia, including, as non-limitingexamples: anemia of chronic disease (ACD), anemia of (e.g., associatedwith) chronic kidney disease (CKD), anemia of cancer, anemia ofinflammation, erythropoiesis stimulating agent (ESA) resistant anemia(for example erythropoietin (EPO) resistant anemia, ESA hyporesponsiveanemia (for example, EPO hyporesponsive anemia), functionaliron-deficiency anemia, and/or iron-restricted anemia.

In a specific embodiment, the present invention provides methods oftreating a BMP6 related disease or disorder by administering to asubject in need thereof an effective amount of the antibodies andantigen-binding fragments thereof of the invention, wherein said diseaseor disorder is anemia. In an embodiment the anemia is anemia of chronicdisease. In an embodiment the chronic disease is chronic kidney disease.In an embodiment the chronic disease is cancer. In an embodiment thechronic disease is inflammation. In an embodiment the anemia (e.g., theanemia of chronic disease) is ESA (for example, EPO)-resistant anemia.In an embodiment the anemia (e.g., the anemia of chronic disease) is ESA(for example, EPO)-hyporesponsive anemia. In an embodiment, the anemia(e.g., the anemia of chronic disease) is iron-restricted anemia. Inembodiments, including in any of the above embodiments, the subject is achronic hemodialysis (HD) subject. In embodiments, including in any ofthe above embodiments, the subject has renal disease, for example,end-stage renal disease.

In a specific embodiment, the present invention provides methods oftreating a BMP6 related disease or disorder by administering to asubject in need thereof an effective amount of an antibody andantigen-binding fragment thereof of the invention, wherein said diseaseor disorder is functional iron deficiency anemia. In an embodiment, thesubject is an ESA (for example, EPO) treated chronic hemodialysispatients. In an embodiment, the subject is an ESA (for example, EPO)treated chronic hemodialysis patient with chronic kidney disease.

In a specific embodiment, the present invention provides methods oftreating anemia by administering to a subject in need thereof aneffective amount of a composition comprising an antibody of the presentinvention. In a specific embodiment, the present invention providesmethods of treating anemia by administering to a subject in need thereofan effective amount of a composition comprising an antibody of thepresent invention. In an embodiment the anemia is anemia of chronickidney disease. In an embodiment the anemia is ESA (for example,EPO)-resistant anemia. In an embodiment the anemia is ESA (for example,EPO)-hyporesponsive anemia. In an embodiment, the anemia isiron-restricted anemia. In an embodiment, the anemia is anemiaassociated with kidney disease, for example, chronic kidney disease. Inembodiments, including in any of the above embodiments, the subject is achronic hemodialysis (HD) subject. In embodiments, including in any ofthe above embodiments, the subject has renal disease, for example,end-stage renal disease.

In a specific embodiment, the present invention provides methods oftreating a BMP6 related disease or disorder by administering to asubject in need thereof an effective amount of a composition comprisingan antibody of the present invention, wherein said disease or disorderis functional iron deficiency anemia. In an embodiment, the subject isan ESA (for example, EPO) treated chronic hemodialysis patients. In anembodiment, the subject is an ESA (for example, EPO) treated chronichemodialysis patient with chronic kidney disease.

In a specific embodiment, the present invention provides methods oftreating anemia.

In a specific embodiment, the present invention provides a method forreducing a subject's ESA (for example, EPO) dosing needs byadministering to a subject in need thereof an effective amount of anantibody or antigen-binding fragment thereof of the invention. In anembodiment the anemia is anemia of chronic disease. In an embodiment thechronic disease is chronic kidney disease. In an embodiment the chronicdisease is cancer. In an embodiment the chronic disease is inflammation.In an embodiment the anemia (e.g., the anemia of chronic disease) is ESA(for example, EPO)-resistant anemia. In an embodiment the anemia (e.g.,the anemia of chronic disease) is ESA (for example, EPO)-hyporesponsiveanemia. In an embodiment, the anemia (e.g, the anemia of chronicdisease) is iron-restricted anemia. In embodiments, including in any ofthe above embodiments, the subject is a chronic hemodialysis (HD)subject. In embodiments, including in any of the above embodiments, thesubject has renal disease, for example, end-stage renal disease. Inembodiments, the methods and use of the invention result in a decreasein a patients ESA resistance index (ERI).

In a specific embodiment, the present invention provides methods ofreducing s subject's ESA (for example, EPO) dosing needs byadministering to a subject in need thereof an effective amount of anantibody or antigen-binding fragment thereof of the invention, whereinsaid subject has functional iron deficiency anemia. In an embodiment,the subject is an ESA (for example, EPO) treated chronic hemodialysispatient. In an embodiment, the subject is an ESA (for example, EPO)treated chronic hemodialysis patient with chronic kidney disease.

In a specific embodiment, the present invention provides a method forreducing a subject's ESA (for example, EPO) dosing needs byadministering to a subject in need thereof an effective amount of acomposition comprising an antibody of the present invention. In anembodiment, the subject has anemia. In an embodiment the anemia isanemia of chronic disease. In an embodiment the chronic disease ischronic kidney disease. In an embodiment the chronic disease is cancer.In an embodiment the chronic disease is inflammation. In an embodimentthe anemia (e.g., the anemia of chronic disease) is ESA (for example,EPO)-resistant anemia. In an embodiment the anemia (e.g., the anemia ofchronic disease) is ESA (for example, EPO)-hyporesponsive anemia. In anembodiment, the anemia (e.g., the anemia of chronic disease) isiron-restricted anemia. In embodiments, including in any of the aboveembodiments, the subject is a chronic hemodialysis (HD) subject. Inembodiments, including in any of the above embodiments, the subject hasrenal disease, for example, end-stage renal disease.

In a specific embodiment, the present invention provides methods ofreducing s subject's ESA (for example, EPO) dosing needs byadministering to a subject in need thereof an effective amount of acomposition comprising an antibody of the present invention, whereinsaid subject has functional iron deficiency anemia. In an embodiment,the subject is an ESA (for example, EPO) treated chronic hemodialysispatient. In an embodiment, the subject is an ESA (for example, EPO)treated chronic hemodialysis patient with chronic kidney disease.

In a specific embodiment, the present invention provides a method forreducing a subject's iron (for example, IV iron) dosing needs byadministering to a subject in need thereof an effective amount of theantibodies and antigen-binding fragments thereof of the invention. In anembodiment, the subject has anemia. In an embodiment the anemia isanemia of chronic disease. In an embodiment the chronic disease ischronic kidney disease. In an embodiment the chronic disease is cancer.In an embodiment the chronic disease is inflammation. In an embodimentthe anemia (e.g., the anemia of chronic disease) is ESA (for example,EPO)-resistant anemia. In an embodiment the anemia (e.g., the anemia ofchronic disease) is ESA (for example, EPO)-hyporesponsive anemia. In anembodiment, the anemia (e.g., the anemia of chronic disease) isiron-restricted anemia. In embodiments, including in any of the aboveembodiments, the subject is a chronic hemodialysis (HD) subject. Inembodiments, including in any of the above embodiments, the subject hasrenal disease, for example, end-stage renal disease.

In a specific embodiment, the present invention provides methods ofreducing s subject's iron (for example, IV iron) dosing needs byadministering to a subject in need thereof an effective amount of theantibodies and antigen-binding fragments thereof of the invention,wherein said subject has functional iron deficiency anemia. In anembodiment, the subject is an ESA (for example, EPO) treated chronichemodialysis patient. In an embodiment, the subject is an ESA (forexample, EPO) treated chronic hemodialysis patient with chronic kidneydisease.

In a specific embodiment, the present invention provides a method forreducing a subject's iron (for example, IV iron) dosing needs byadministering to a subject in need thereof an effective amount of acomposition comprising an antibody of the present invention. In anembodiment, the subject has anemia. In an embodiment the anemia isanemia of chronic disease. In an embodiment the chronic disease ischronic kidney disease. In an embodiment the chronic disease is cancer.In an embodiment the chronic disease is inflammation. In an embodimentthe anemia (e.g., the anemia of chronic disease) is ESA (for example,EPO)-resistant anemia. In an embodiment the anemia (e.g., the anemia ofchronic disease) is ESA (for example, EPO)-hyporesponsive anemia. In anembodiment, the anemia (e.g., the anemia of chronic disease) isiron-restricted anemia. In embodiments, including in any of the aboveembodiments, the subject is a chronic hemodialysis (HD) subject. Inembodiments, including in any of the above embodiments, the subject hasrenal disease, for example, end-stage renal disease.

In a specific embodiment, the present invention provides methods ofreducing s subject's iron (for example, IV iron) dosing needs byadministering to a subject in need thereof an effective amount of acomposition comprising an antibody of the present invention, whereinsaid subject has functional iron deficiency anemia. In an embodiment,the subject is an ESA (for example, EPO) treated chronic hemodialysispatient. In an embodiment, the subject is an ESA (for example, EPO)treated chronic hemodialysis patient with chronic kidney disease.

In a specific embodiment, the invention provides a method for reducing asubject's iron (for example, IV iron) dosing needs and reducing asubject's ESA (for example, EPO) dosing needs, comprising administeringthe antibody or antigen binding fragment of the invention or acomposition comprising said antibody or antigen binding fragment. In anembodiment the anemia is anemia of chronic disease. In an embodiment thechronic disease is chronic kidney disease. In an embodiment the chronicdisease is cancer. In an embodiment, the subject has anemia. In anembodiment the chronic disease is inflammation. In an embodiment theanemia (e.g., the anemia of chronic disease) is ESA (for example,EPO)-resistant anemia. In an embodiment the anemia (e.g., the anemia ofchronic disease) is ESA (for example, EPO)-hyporesponsive anemia. In anembodiment, the anemia (e.g., the anemia of chronic disease) isiron-restricted anemia. In embodiments, including in any of the aboveembodiments, the subject is a chronic hemodialysis (HD) subject. Inembodiments, including in any of the above embodiments, the subject hasrenal disease, for example, end-stage renal disease.

In an embodiment, present invention provides methods of mobilizingsequestered iron by administering to a subject in need thereof aneffective amount of the antibodies and antigen-binding fragments thereofof the invention. In an embodiment, present invention provides methodsof mobilizing sequestered iron by administering to a subject in needthereof an effective amount of a composition comprising an antibody ofthe present invention.

In an embodiment, the present invention provides a method for improving(for example, increasing) the level of hemoglobin in a subject withanemia, while reducing the need for dosing with erythropoietin and/oriron (e.g., IV iron), said method comprising administering to a subjectin need thereof an antibody or antigen binding fragment thereof of theinvention. In an embodiment, the anemia is anemia associated withchronic disease. In an embodiment, the improving the level of hemoglobincomprises improving the level to a level as specified by a clinicalpractice guideline, for example, Kidney Disease: Improving GlobalOutcomes (KDIGO) Anemia Work Group. KDIGO Clinical Practice Guidelinefor Anemia in Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2:279-335, the contents of which are hereby incorporated by reference intheir entirety. In an embodiment, the improving the level of hemoglobincomprises improving the level of hemoglobin to at least about 11.0 g/dL,e.g., to from about 11.0 g/dL to about 12.5 g/dL. In an embodiment, theimproving the level of hemoglobin comprises improving the level ofhemoglobin to at least 11.0 g/dL, e.g., to from 11.0 g/dL to 12.5 g/dL.

In an embodiment, the present invention provides a method for improving(for example, increasing) the level of hemoglobin in a subject withanemia, while reducing the need for dosing with erythropoietin and/oriron e.g., IV iron), said method comprising administering to a subjectin need thereof a composition comprising an antibody of the invention.In an embodiment, the anemia is anemia associated with chronic disease.In an embodiment, the improving the level of hemoglobin comprisesimproving the level to a level as specified by a clinical practiceguideline, for example, Kidney Disease: Improving Global Outcomes(KDIGO) Anemia Work Group. KDIGO Clinical Practice Guideline for Anemiain Chronic Kidney Disease. Kidney inter., Suppl. 2012; 2: 279-335, thecontents of which are hereby incorporated by reference in its entirety.In an embodiment, the improving the level of hemoglobin comprisesimproving the level of hemoglobin to at least about 11.0 g/dL, e.g., tofrom about 11.0 g/dL to about 12.5 g/dL. In an embodiment, the improvingthe level of hemoglobin comprises improving the level of hemoglobin toat least 11.0 g/dL, e.g., to from 11.0 g/dL to 12.5 g/dL.

In an embodiment, the present invention provides a method formaintaining the level of hemoglobin in a subject with anemia, whilereducing the need for dosing with erythropoietin and/or iron (e.g., IViron), said method comprising administering to a subject in need thereofan antibody or antigen binding fragment thereof of the invention. In anembodiment, the anemia is anemia associated with chronic disease. In anembodiment, the improving the level of hemoglobin comprises improvingthe level to a level as specified by a clinical practice guideline, forexample, Kidney Disease: Improving Global Outcomes (KDIGO) Anemia WorkGroup. KDIGO Clinical Practice Guideline for Anemia in Chronic KidneyDisease, Kidney inter., Suppl. 2012; 2: 279-335, the contents of whichare hereby incorporated by reference in its entirety. In an embodiment,the improving the level of hemoglobin comprises improving the level ofhemoglobin to at least about 11.0 g/dL, e.g., to from about 11.0 g/dL toabout 12.5 g/dL. In an embodiment, the improving the level of hemoglobincomprises improving the level of hemoglobin to at least 11.0 g/dL, e.g.,to from 11.0 g/dL to 12.5 g/dL.

In an embodiment, the present invention provides a method formaintaining the level of hemoglobin in a subject with anemia, whilereducing the need for dosing with erythropoietin and/or iron (e.g., IViron), said method comprising administering to a subject in need thereofa composition comprising an antibody of the invention. In an embodiment,the anemia is anemia associated with chronic disease. In an embodiment,the improving the level of hemoglobin comprises improving the level to alevel as specified by a clinical practice guideline, for example, KidneyDisease: Improving Global Outcomes (KDIGO) Anemia Work Group. KDIGOClinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidneyinter., Suppl. 2012; 2: 279-335, the contents of which are herebyincorporated by reference in its entirety. In an embodiment, theimproving the level of hemoglobin comprises improving the level ofhemoglobin to at least about 11.0 g/dL, e.g., to from about 11.0 g/dL toabout 12.5 g/dL. In an embodiment, the improving the level of hemoglobincomprises improving the level of hemoglobin to at least 11.0 g/dL, e.g.,to from 11.0 g/dL, to 12.5 g/dL.

In one embodiment, the isolated antibody or antigen-binding fragmentthereof described in Table 1 and/or Table 14 can be administered to apatient in need thereof in conjunction with a therapeutic method orprocedure, such as described herein or known in the art. Such a methodor procedure includes, as non-limiting examples: administration of atherapeutically effective amount of ESA (for example, EPO),erythropoietin, or iron, and blood transfusion. Treatment is typicallycontinued at intervals for a period of a week, a month, three months,six months or a year. In some patients, treatment is administered for upto the rest of a patient's life.

When the therapeutic agents of the present invention are administeredtogether with another agent, the two can be administered sequentially ineither order or simultaneously. In some aspects, an antibody of thepresent invention is administered to a subject who is also receivingtherapy with a second agent or method (e.g., ESA, erythropoietin, iron,blood transfusion). In other aspects, the binding molecule isadministered in conjunction with surgical treatments.

Suitable agents for combination treatment with BMP6-binding antibodiesinclude agents known in the art that inhibit or reduce the expression,level, stability and/or activity of BMP6. Such agents includeantibodies, siRNAs, and small molecules to BMP6.

-   Various antibodies to BMP6 are known. in the art, including, inter    alia, those described in:-   Andriopoulos et al. 2009 Nat. Genet. 41: 482-487;-   Arndt et at. 2010 Gastroent. 138: 372-382;-   Barnes et al. 1995 World J. Urol, 13: 337-343;-   Camaschella et al. 2009 Nat. Genet. 41: 386-388;-   Celement et al. 1999 Int. J. Cancer 80: 250-256;-   Corradini et at. 2011 Hepatol. 54: 273-284;-   Crews et al. 2010 J. Neuro. 30: 12252-12262;-   Dai et al. 2005 Cancer Res. 65: 8274;-   Darby et al. 2007 J. Pathol. 214: 394-404;-   Hadziahmetovic et al. 2011 179: 335-348;-   Hamdy et al. 1997 Cancer Res. 57: 4427;-   Haudenschild et al. 2004 Cancer Res. 64: 8276;-   Hee et al. 2008 J. Orth. Res. 27: 162-168;-   Herrera et al. 2009 BMC Cell Biol. 10: 20;-   Inagaki et al. 2005 Endocrin. 147: 2681-2689;-   Jung et al. 2008 Stem Cells 26: 2042-2051;-   Kaiser et al. 1998 J. Invest. Derm. 111: 1145-1152;-   Kautz et al. 2011 Haematol. 96: 199-203;-   Khalaf et al. 2012 Eur. J. Endocrin, 168: 437-444;-   Kochanowska et al. 2002 Exp. Biol. Med. 227: 57-62;-   Li et al. 2006: Int. J. Med. Sci. 3: 97-105;-   Meynard et al. 2011 Blood 118: 747-756;-   Pederson et al. 2008 Proc. Natl. Acad. Sci. USA 105: 20764-69;-   Plant et al. 2002 J. Bone Min. Res. 17: 782-790;-   Schluesener et al. 1994 Atheroscl. 113: 153-156;-   Schluesener et al. 2004 GLIA 12: 161-164;-   Shi et al. 2009 Fert. Steril. 92: 1794-1798;-   Varley et al. 1996 Exp. Neur. 140: 84-94;-   Wang et al. 2007 Mol. Cell. Neurosci. 34: 653-661; and-   Zhang et al. 2006 Neurosci. 138: 47-53;-   U.S. Pat. No. 8,795,665; and-   WO 2010/056981;-   Additional antibodies to BMP6 are known in the art; many are    commercially available.-   Various siRNAs to BMP6 are known in the art, including, inter alia,    those described in:-   He et al. 2003 Cell. Signal. 25: 1372-1378;-   Ikeda et al. 2012 PLoS 0040465;-   Kautz et al. 2008 Blood 112: 1503;-   Mi et al. 2011 J. Cancer Res. Clin. Oncol. 137: 245;-   Xia et al. 2007 J. Biol. Chem. 282: 18129-18140;-   Xia et al. 2008 Blood 111: 5195; and-   Yang et al. 2009 Int. J. Bioch. Cell Biol. 41: 853-861.

Additional inhibitors of BMP6 are known. Any of these can be used incombination with any antibody or antigen-binding fragment thereofdisclosed herein.

A combination therapy regimen may be additive, or it may producesynergistic results (e.g., reductions in BMP6 activity more thanexpected for the combined use of the two agents). In one embodiment, thepresent invention provide a combination therapy for preventing and/ortreating anemia or another BMP6 related disease as described above withBMP6-binding antibody of the invention and an anti-anemia agent ormethod, such as ESA, erythropoietin, iron, or blood transfusion.

Diagnostic Uses

In one aspect, the invention encompasses diagnostic assays fordetermining BMP6 and/or nucleic acid expression as well as BMP6function, in the context of a biological sample (e.g., blood, serum,cells, tissue) or from individual is afflicted with a disease ordisorder, or is at risk of developing a disorder associated with anemia.Diagnostic assays, such as competitive assays rely on the ability of alabelled analogue (the “tracer”) to compete with the test sample analytefor a limited number of binding sites on a common binding partner. Thebinding partner generally is insolubilized before or after thecompetition and then the tracer and analyte bound to the binding partnerare separated from the unbound tracer and analyte. This separation isaccomplished by decanting (where the binding partner waspreinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsin order to quantitatively determine the amount of analyte present inthe test sample. These assays are called ELISA systems when enzymes areused as the detectable markers. In an assay of this form, competitivebinding between antibodies and BMP6-binding antibodies results in thebound BMP6, preferably the BMP6 epitopes of the invention, being ameasure of antibodies in the serum sample, most particularly,neutralising antibodies in the serum sample.

A significant advantage of the assay is that measurement is made ofneutralising antibodies directly (i.e., those which interfere withbinding of BMP6, specifically, epitopes). Such an assay, particularly inthe form of an ELISA test has considerable applications in the clinicalenvironment and in routine blood screening. In the clinical diagnosis ormonitoring of patients with disorders associated with anemia, thedetection of BMP6 proteins in comparison to the levels in acorresponding biological sample from a normal subject is indicative of apatient with disorders associated with anemia.

In vivo diagnostic or imaging is described in US2006/0067935. Briefly,these methods generally comprise administering or introducing to apatient a diagnostically effective amount of BMP6 binding molecule thatis operatively attached to a marker or label that is detectable bynon-invasive methods. The antibody-marker conjugate is allowedsufficient time to localize and bind to BMP6. The patient is thenexposed to a detection device to identify the detectable marker, thusforming an image of the location of the BMP6 binding molecules in thetissue of a patient. The presence of BMP6 binding antibody or anantigen-binding fragment thereof is detected by determining whether anantibody-marker binds to a component of the tissue. Detection of anincreased level in BMP6 proteins or a combination of protein incomparison to a normal individual without anemia is indicative of apredisposition for and/or on set of disorders associated with anemia.These aspects of the invention are also for use in tissue imagingmethods and combined diagnostic and treatment methods.

The invention also pertains to the field of predictive medicine in whichdiagnostic assays, prognostic assays, pharmacogenomics, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual prophylactically.

The invention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with dysregulation of BMP6 pathway activity. For example,mutations in BMP6 gene can be assayed in a biological sample. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with BMP6, nucleic acid expression oractivity.

Another aspect of the invention provides methods for determining BMP6nucleic acid expression or BMP6 activity in an individual to therebyselect appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as “pharmacogenomics”). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention provides a method of monitoring theinfluence of agents (e.g., drugs) on the expression or activity of BMP6in clinical trials.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising theBMP6-binding antibody or binding fragment thereof formulated togetherwith a pharmaceutically acceptable carrier. The compositions canadditionally contain one or more other therapeutical agents that aresuitable for treating or preventing a BMP6-associated disease (e.g.,anemia). Pharmaceutically carriers enhance or stabilize the composition,or to facilitate preparation of the composition. Pharmaceuticallyacceptable carriers include solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible.

A pharmaceutical composition of the present invention can beadministered by a variety of methods known in the art. The route and/ormode of administration vary depending upon the desired results.Administration can be intravenous, intramuscular, intraperitoneal, orsubcutaneous, or administered proximal to the site of the target. Thepharmaceutically acceptable carrier should be suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound, i.e., antibody, bispecific andmultispecific molecule, may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

The composition should be sterile and fluid. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin. Pharmaceutical compositions of the inventioncan be prepared in accordance with methods well known and routinelypracticed in the art. See, e.g., Remington: The Science and Practice ofPharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978. Pharmaceutical compositions are preferablymanufactured under GMP conditions. Typically, a therapeuticallyeffective dose or efficacious dose of the BMP6-binding antibody isemployed in the pharmaceutical compositions of the invention. TheBMP6-binding antibodies are formulated into pharmaceutically acceptabledosage forms by conventional methods known to those of skill in the art.Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors.

A physician or veterinarian can start doses of the antibodies andantigen-binding fragments thereof of the invention employed in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and gradually increase the dosage untilthe desired effect is achieved. In general, effective doses of thecompositions of the present invention, for the treatment of an allergicinflammatory disorder described herein vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Treatment dosages need to be titrated tooptimize safety and efficacy. For systemic administration with anantibody, the dosage ranges from about 0.0001 to 100 mg/kg, and moreusually 0.001 to 15 mg/kg, of the host body weight. An exemplarytreatment regime entails systemic administration (e.g., intravenous orsubcutaneous) once, or alternatively, more than once on a dosingschedule (e.g., repeat dosing), for example, once per every two weeks,once every three weeks, or once a month or once every 3 to 6 months. Forintravitreal administration with an antibody, the dosage ranges fromabout 0.0001 to about 10 mg. An exemplary treatment regime entailssystemic administration once per every two weeks, once every threeweeks, or once a month or once every 3 to 6 months.

Antibody is usually administered on multiple occasions. intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of BMP6-bindingantibody in the patient. In some methods of systemic administration,dosage is adjusted to achieve a plasma antibody concentration of 1-1000□g/ml and in some methods 25-500 □g/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanizedantibodies show longer half life than that of chimeric antibodies andnonhuman antibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

In a specific embodiment the composition comprising the antibody orantigen binding fragment of the invention is administered at a dose(antibody or antigen-binding fragment thereof) of between 0.001 mg/kgand 0.1 mg/kg. In a specific embodiment the composition comprising theantibody or antigen binding fragment of the invention is administered ata dose of between about 0.001 mg/kg and about 0.1 mg/kg. In a specificembodiment the composition comprising the antibody or antigen bindingfragment of the invention is administered at a dose of 0.001 mg/kg,0.0016 mg/kg, 0.0025 mg/kg, 0.0040 mg/kg, 0.0063 mg/kg, 0.01 mg/kg,0.016 mg/kg, 0.025 mg/kg, 0.040 mg/kg, 0.063 mg/kg, or 0.1 mg/kg. In aspecific embodiment the composition comprising the antibody or antigenbinding fragment of the invention is administered at a dose of about0.001 mg/kg, about 0.0016 mg/kg, about 0.0025 mg/kg, about 0.0040 mg/kg,about 0.0063 mg/kg, about 0.01 mg/kg, about 0.016 mg/kg, about 0.025mg/kg, about 0.040 mg/kg, about 0.063 mg/kg, or about 0.1 mg/kg. In anembodiment, the composition comprising the antibody or antigen bindingfragment of the invention is administered, including, for example, atany of the doses recited above, intravenously. In embodiments, theintravenous administration is an intravenous infusion. In embodiments,the infusion takes place over 30-60 minutes. In embodiments, theinfusion takes place over about 30-60 minutes.

Level of Ferritin

The disclosed methods may involve the determination of the level offerritin from a biological sample, e.g., blood or serum, and inembodiments, patients, e.g., patients having a disease described herein,e.g., anemia, are selected for treatment with a BMP6 antagonist (e.g.,as described herein, e.g., a BMP6 antibody, e.g., Antibody 7, e.g., asdescribed in Table 1) or are predicted to have a response to BMP6antagonist therapy based on the level of ferritin.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is ≤about 1500 ng/mL. In embodiments, the level of ferritin, e.g., thelevel of ferritin predictive of response (e.g., increased response) totreatment with BMP6 is ≤1500 ng/mL. In embodiments, the level offerritin is <1500 ng/mL. In embodiments, the level of ferritin is ≤about2000 ng/mL. In embodiments, the level of ferritin is ≤2000 ng/mL. Inembodiments, the level of ferritin is <about 2000 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is ≤about 1450 ng/mL, e.g., ≤about 1400 ng/mL, ≤about 1350 ng/mL, ≤about1300 ng/mL, ≤about 1250 ng/mL, ≤about 1200 ng/mL, ≤about 1150 ng/mL,≤about 1100 ng/mL, ≤about 1050 ng/mL, or ≤about 1000 ng/mL, etc.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is ≥about 200 ng/mL, e.g., ≥about 250 ng/mL, ≥about 300 ng/mL, ≥about350 ng/mL, ≥about 400 ng/mL, ≥about 450 ng/mL, ≥about 500 ng/mL, ≥about550 ng/mL, ≥about 600 ng/mL, ≥about 650 ng/mL, ≥about 700 ng/mL, ≥about750 ng/mL, ≥about 800 ng/mL, ≥about 850 ng/mL, ≥about 900 ng/mL, ≥about950 ng/mL, ≥about 1000 ng/mL, or ≥about 1500 ng/mL, etc. In embodiments,the level of ferritin is between any of the levels recited above andabout 2000 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is ≥about 500 ng/mL, ≥about 550 ng/mL, ≥about 600 ng/mL, ≥about 650ng/mL, ≥about 700 ng/mL, ≥about 750 ng/mL, ≥about 800 ng/mL, ≥about 850ng/mL, ≥about 900 ng/mL, ≥about 950 ng/mL, ≥about 1000 ng/mL, or ≥about1500 ng/mL, etc. In embodiments, the level of ferritin is between any ofthe levels recited above and about 2000 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is ≥about 500 ng/mL. Inembodiments, the level of ferritin is between about 500 ng/mL, and about2000 ng/mL. In embodiments, the level of ferritin is between about 500ng/mL, and about 1000 ng/mL. In embodiments, the level of ferritin isbetween about 500 ng/mL and about 1500 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 200 ng/mL and about 1500 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 300ng/mL and about 1500 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 400 ng/mL and about 1500 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 500 ng/mL and about 1500 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 600ng/mL, and about 1500 ng/mL. In embodiments, the level of ferritin,e.g., the level of ferritin predictive of response (e.g., increasedresponse) to treatment with BMP6 is between about 700 ng/mL and about1500 ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between about 800 ng/mL and about 1500 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 900 ng/mL and about 1500 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1000and about 1500 ng/mL. In embodiments, the level of ferritin, e.g., thelevel of ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between about 1100 ng/mL and about 1500 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response increased response) to treatment with BMP6 isbetween about 1200 ng/mL, and about 1500 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1300ng/mL and about 1500 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1400 ng/mL and about 1500 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 200 ng/mL and about 1600 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 300ng/mL and about 1600 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 400 ng/mL and about 1600 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 500 ng/mL and about 1600 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 600ng/mL, and about 1600 ng/mL. In embodiments, the level of ferritin,e.g., the level of ferritin predictive of response (e.g., increasedresponse) to treatment with BMP6 is between about 700 ng/mL and about1600 ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between about 800 ng/mL and about 1600 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 900 ng/mL and about 1600 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1000ng/mL and about 1600 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1100 ng/mL and about 1600 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1200 ng/mL, and about 1600 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1300ng/mL and about 1600 ng/mL. In embodiments, the level of ferritin, thelevel of ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between about 1400 ng/mL and about 1600 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1500 ng/mL and about 1600 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 200 ng/mL and about 1700 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 300ng/mL and about 1700 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 400 ng/mL and about 1700 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 500 ng/mL and about 1700 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 600ng/mL and about 1700 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 700 ng/mL and about 1700 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 800 ng/mL and about 1700 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 900ng/mL and about 1700 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1000 ng/mL and about 1700 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1100 ng/mL and about 1700 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1200ng/mL and about 1700 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g, increased response)to treatment with BMP6 is between about 1300 ng/mL and about 1700 ng/mL.In embodiments, the level of ferritin, the level of ferritin predictiveof response (e.g., increased response) to treatment with BMP6 is betweenabout 1400 ng/mL and about 1700 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BNIP6 is between about 1500 ng/mLand about 1700 ng/mL. In embodiments, the level of ferritin, e.g., thelevel of ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between about 1600 ng/mL and about 1700 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 200 ng/mL and about 1800 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 300ng/mL and about 1800 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 400 ng/mL and about 1800 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response increased response) to treatment with BMP6 isbetween about 500 ng/mL and about 1800 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between about 600 ng/mLand about 1800 ng/mL. In embodiments, the level of ferritin, e.g., thelevel of ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between about 700 ng/mL and about 1800 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 800 ng/mL and about 1800 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 900ng/mL and about 1800 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1000 ng/mL and about 1800 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1100 ng/mL and about 1800 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1200ng/mL, and about 1800 ng/mL. In embodiments, the level of ferritin,e.g., the level of ferritin predictive of response (e.g., increasedresponse) to treatment with BMP6 is between about 1300 ng/mL and about1800 ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between about 1400 ng/mL and about 1800 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1500 ng/mL and about 1800 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1600ng/mL and about 1800 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1700 ng/mL and about 1800 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 200 ng/mL and about 1900 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 300ng/mL and about 1900 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 400 ng/mL and about 1900 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 500 ng/mL and about 1900 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 600ng/mL and about 1900 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 700 ng/mL and about 1900 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 800 ng/mL and about 1900 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 900ng/mL and about 1900 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1000 ng/mL and about 1900 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment withBNIP6 is between about 1100 ng/mL and about 1900 ng/mL. In embodiments,the level of ferritin, e.g., the level of ferritin predictive ofresponse (e.g., increased response) to treatment with BMP6 is betweenabout 1200 ng/mL and about 1900 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between about 1300 ng/mLand about 1900 ng/mL. In embodiments, the level of ferritin, e.g., thelevel of ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between about 1400 ng/mL and about 1900 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1500 ng/mL and about 1900 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1600ng/mL and about 1900 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1700 ng/mL and about 1900 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1800 ng/mL and about 1900 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 200 ng/mL and about 2000 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 300ng/mL and about 2000 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 400 ng/mL and about 2000 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 500 ng/mL and about 2000 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 600ng/mL and about 2000 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 700 ng/mL and about 2000 ng/mL.In embodiments, the level of terrain, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 800 ng/mL and about 2000 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 900ng/mL and about 2000 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1000 ng/mL and about 2000 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1100 ng/mL and about 2000 ng/mL. In embodiments, thelevel of terrain, e.g., the level of terrain predictive of response(e.g., increased response) to treatment with BMP6 is between about 1200ng/mL and about 2000 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1300 ng/mL and about 2000 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1400 ng/mL and about 2000 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between about 1500ng/mL and about 2000 ng/mL. In embodiments, the level of ferritin, e.g.,the level of ferritin predictive of response (e.g., increased response)to treatment with BMP6 is between about 1600 ng/mL and about 2000 ng/mL.In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between about 1700 ng/mL and about 2000 ng/mL. In embodiments, thelevel of terrain, e.g., the level of terrain predictive of response(e.g., increased response) to treatment with BMP6 is between about 1800ng/mL and about 2000 ng/mL. In embodiments, the level of terrain, e.g.,the level of terrain predictive of response (e.g., increased response)to treatment with BMP6 is between about 1900 ng/mL and about 2000 ng/mL.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 200 ng/mL and 1500 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 300 ng/mL and 1500ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 400 ng/mL and 1500 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 500 ng/mL and 1500ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 600 ng/mL and 1500 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of responseincreased response) to treatment with BMP6 is between 700 ng/mL and 1500ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 800 ng/mL and 1500 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 900 ng/mL and 1500ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1000 ng/mL and 1500 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1100 ng/mLand 1500 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1200 ng/mL and 1500 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1300 ng/mL and 1500 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 1400 ng/mL and1500 ng/mL. In preferred embodiments, all ranges are inclusive of therecited endpoint values.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 200 ng/mL and 1600 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 300 ng/mL and 1600ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 400 ng/mL and 1600 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 500 ng/mL and 1600ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 600 ng/mL and 1600 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 700 ng/mL and 1600ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 800 ng/mL and 1600 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 900 ng/mL and 1600ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1000 ng/mL and 1600 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1100 ng/mLand 1600 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1200 ng/mL and 1600 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1300 ng/mL and 1600 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) treatment with BMP6 is between 1400 ng/mL and 1600ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1500 ng/mL and 1600 ng/mL. In preferredembodiments, all ranges are inclusive of the recited endpoint values. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 200 ng/mL and 1700 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 300 ng/mL and 1700ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 400 ng/mL and 1700 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 500 ng/mL and 1700ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 600 ng/mL and 1700 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 700 ng/mL and 1700ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g, increased response) to treatmentwith BMP6 is between 800 ng/mL and 1700 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 900 ng/mL and 1700ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1000 ng/mL and 1700 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1100 ng/mLand 1700 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1200 ng/mL and 1700 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1300 ng/mL and 1700 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 1400 ng/mL and1700 ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1500 ng/mL and 1700 ng/mL. In embodiments, thelevel of ferritin, the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 1600 ng/mL and1700 ng/mL. In preferred embodiments, all ranges are inclusive of therecited endpoint values.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 200 ng/mL and 1800 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 300 ng/mL and 1800ng/mL. In embodiments, the level of ferritin, the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 400 ng/mL and 1800 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 500 ng/mL and 1800ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 600 ng/mL and 1800 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 700 ng/mL and 1800ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 800 ng/mL and 1800 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 900 ng/mL and 1800ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1000 ng/mL and 1800 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1100 ng/mLand 1800 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response increased response) to treatment withBMP6 is between 1200 ng/mL and 1800 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 1300 ng/mL and1800 ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1400 ng/mL and 1800 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1500 ng/mLand 1800 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1600 ng/mL and 1800 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1700 ng/mL and 1800 ng/mL. In preferred embodiments, allranges are inclusive of the recited endpoint values.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 200 ng/mL and 1900 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 300 ng/mL and 1900ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 400 ng/mL and 1900 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 500 ng/mL and 1900ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatment:with BMP6 is between 600 ng/mL and 1900 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 700 ng/mL and 1900ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 800 ng/mL and 1900 ng/mL. In embodiments, the levelof ferritin, the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 900 ng/mL and 1900ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1000 ng/mL and 1900 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1100 ng/mLand 1900 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1200 ng/mL and 1900 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1300 ng/mL and 1900 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 1400 ng/mL and1900 ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1500 ng/mL and 1900 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1600 ng/mLand 1900 ng/mL. in embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1700 ng/mL and 1900 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1800 ng/mL and 1900 ng/mL. In preferred embodiments, allranges are inclusive of the recited endpoint values.

In embodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 200 ng/mL and 2000 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 300 ng/mL and 2000ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 400 ng/mL and 2000 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 500 ng/mL and 2000ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 600 ng/mL and 2000 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 700 ng/mL and 2000ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 800 ng/mL and 2000 ng/mL. In embodiments, the levelof ferritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 900 ng/mL and 2000ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1000 ng/mL and 2000 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1100 ng/mLand 2000 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1200 ng/mL and 2000 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1300 ng/mL and 2000 ng/mL. In embodiments, the level offerritin, e.g., the level of ferritin predictive of response (e.g.,increased response) to treatment with BMP6 is between 1400 ng/mL and2000 ng/mL. In embodiments, the level of ferritin, e.g., the level offerritin predictive of response (e.g., increased response) to treatmentwith BMP6 is between 1500 ng/mL and 2000 ng/mL. In embodiments, thelevel of ferritin, e.g., the level of ferritin predictive of response(e.g., increased response) to treatment with BMP6 is between 1600 ng/mLand 2000 ng/mL. In embodiments, the level of ferritin, e.g., the levelof ferritin predictive of response (e.g., increased response) totreatment with BMP6 is between 1700 ng/mL and 2000 ng/mL. Inembodiments, the level of ferritin, e.g., the level of ferritinpredictive of response (e.g., increased response) to treatment with BMP6is between 1800 ng/mL and 2000 ng/mL. In preferred embodiments, allranges are inclusive of the recited endpoint values.

In embodiments, the levels of ferritin described above are measured froma biological sample selected from blood and serum. In embodiments, thelevels of ferritin described above are measured from serum.

Techniques for Assaying, Diagnostic Methods and Methods of ProducingTransmittable Form of Information

The disclosed methods are useful for the treatment, prevention, oramelioration of diseases associated with low iron levels, e.g., diseasesdescribed herein, e.g., anemia, as well as predicting the likelihood ofa disease patient's response to treatment with a BMP6 antagonist, e.g.,Antibody 7 (e.g., as described in Table 1). These methods employ, interalia, determining whether a patient has a particular level of ferritin,e.g., as described herein, in a sample from the patient. A biologicalsample from the patient may be assayed for the level of ferritin by anyapplicable conventional means.

Numerous biological samples may be used to identify the presence themarker, e.g., proteins, the level of expression of genes or proteins,and the activity of a protein, e.g., blood, synovial fluid, huffy coat,serum, plasma, lymph, feces, urine, tear, saliva, cerebrospinal fluid,buccal swabs, sputum, or tissue. Various sources within a biologicalsample may be used in the disclosed methods, e.g, one may assay abiological sample, e.g., blood or serum, from the patient for the levelof ferritin. We have determined that the level of ferritin may be auseful biomarker in determining or predicting response to therapy with aBMP9 antagonist. Accordingly, a skilled artisan will understand that onemay identify whether a subject has a given level of ferritin by assayinga biological sample, e.g., blood or serum, from the patient for thelevel of ferritin. In preferred embodiments, patient serum is analyzedto determine whether a subject has a particular level of ferritin. Inother preferred embodiments, patient blood is analyzed to determinewhether a subject has a particular level of ferritin.

As described herein, the invention is based in part on the conclusionthat the level of ferritin may be useful to predict improved response toBMP6 antagonism (e.g., Antibody 7, as described in Table 1) for anemiaor other disease described herein. Detection of ferritin can beperformed using any known method in the art including, but not limited,to immunocytochemical staining, ELISA, flow cytometry, Western blot,spectrophotometry, HPLC, and mass spectrometry. One method for detectingpolypeptide products in a sample is by means of a probe that is abinding protein capable of interacting specifically with a markerprotein (e.g., an antibody capable of binding ferritin protein).Preferably, labeled antibodies, binding portions thereof, or otherbinding partners can be used. The antibodies can be monoclonal orpolyclonal in origin, or may be biosynthetically produced. The bindingpartners may also be naturally occurring molecules or syntheticallyproduced. The amount of complexed proteins is determined using standardprotein detection methodologies described in the art. A detailed reviewof immunological assay design, theory and protocols can be found innumerous texts in the art, including Practical Immunology, Butt, W. R.,ed., Marcel Dekker, New York, 1984. A variety of assays are availablefor detecting proteins with labeled antibodies. Direct labels includefluorescent or luminescent tags, metals, dyes. radionucleides, and thelike, attached to the antibody. Indirect labels include various enzymeswell known in the art, such as alkaline phosphatase, hydrogen peroxidaseand the like. In a one-step assay, polypeptide products, if present, areimmobilized and incubated with a labeled antibody. The labeled antibodybinds to the immobilized target molecule. After washing to removeunbound molecules, the sample is assayed for the label.

The use of immobilized antibodies specific for the proteins orpolypeptides is also contemplated by the present disclosure. Theantibodies can be immobilized onto a variety of solid supports, such asmagnetic or chromatographic matrix particles, the surface of an assayplace (such as microtiter wells), pieces of a solid substrate material(such as plastic, nylon, paper), and the like. An assay strip can beprepared by coating the antibody or a plurality of antibodies in anarray on solid support. This strip can then be dipped into the testsample and then processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

In a two-step assay, immobilized marker (e.g., ferritin) may beincubated with an unlabeled antibody. The unlabeled antibody complex, ifpresent, is then bound to a second, labeled antibody that is specificfor the unlabeled antibody. The sample is washed and assayed for thepresence of the label. The choice of marker used to label the antibodieswill vary depending upon the application. However, the choice of themarker is readily determinable to one skilled in the art. The antibodiesmay be labeled with a radioactive atom, an enzyme, a chromophoric orfluorescent moiety, or a colorimetric tag. The choice of tagging labelalso will depend on the detection limitations desired. Enzyme assays(ELISAs) typically allow detection of a colored product formed byinteraction of the enzyme-tagged complex with an enzyme substrate. Someexamples of radioactive atoms include 32P, 125I, 3H, and 14P. Someexamples of enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase.Some examples of chromophoric moieties include fluorescein andrhodamine. The antibodies may be conjugated to these labels by methodsknown in the art. For example, enzymes and chromophoric molecules may beconjugated to the antibodies by means of coupling agents, such asdialdehydes, carbodiimides, dimaleimides, and the like. Alternatively,conjugation may occur through a ligand-receptor pair. Some suitableligand-receptor pairs include, for example, biotin-avidin or-streptavidin, and antibody-antigen.

In one aspect, the present disclosure contemplates the use of a sandwichtechnique for detecting polypeptide products in biological samples. Thetechnique requires two antibodies capable of binding the protein ofinterest: e.g., one immobilized onto a solid support and one free insolution, but labeled with some easily detectable chemical compound.Examples of chemical labels that may be used for the second antibodyinclude but are not limited to radioisotopes, fluorescent compounds, andenzymes or other molecules which generate colored or electrochemicallyactive products when exposed to a reactant or enzyme substrate. Whensamples containing polypeptide products are placed in this system, thepolypeptide products hinds to both the immobilized antibody and thelabeled antibody. The result is a “sandwich” immune complex on thesupport's surface. The complexed protein is detected by washing awaynonbound sample components and excess labeled antibody, and measuringthe amount of labeled antibody complexed to protein on the support'ssurface. The sandwich immunoassay is highly specific and very sensitive,provided that labels with good limits of detection are used.

Preferably, the presence of polypeptide products in a sample is detectedby radioimmunoassays or enzyme-linked immunoassays, competitive bindingenzyme-linked immunoassays, dot blot, Western blot, chromatography,preferably high performance liquid chromatography (HPLC), or otherassays known in the art. Specific immunological binding of the antibodyto the protein or polypeptide can be detected directly or indirectly.

Dot blotting is routinely practiced by the skilled artisan to detect adesired protein using an antibody as a probe (Promega Protocols andApplications Guide, Second Edition, 1991, Page 263, PromegaCorporation). Samples are applied to a membrane using a dot blotapparatus. A labeled probe is incubated with the membrane, and thepresence of the protein is detected.

Western blot analysis is well known to the skilled artisan (Sambrook etal., Molecular Cloning, A Laboratory Manual, 1989, Vol. 3, Chapter 18,Cold Spring Harbor Laboratory). In Western blot, the sample is separatedby SDS-PAGE. The gel is transferred to a membrane. The membrane isincubated with labeled antibody for detection of the desired protein.

The assays described above involve steps such as but not limited to,immunoblotting, immunodiffusion, immunoelectrophoresis, orimmunoprecipitation. In some embodiments, an automatic analyzer is usedto determine the presence and/or level of ferritin.

The level of ferritin activity may be assayed by various methodsdisclosed in the art, e.g., via the methods set forth in Kochan et al.(2011) Proc Natl Acad Sci USA. 108(19):7745-50.

For comparative purposes, the level of ferritin expression, ferritinprotein, or ferritin activity from a patient may be compared to thelevel of ferritin expression, ferritin protein, or ferritin activityfrom a control. The control may be a reference level of ferritinexpression, ferritin protein, or ferritin activity derived from subjects(e.g., anemia patients) known to respond well to treatment with a BMP6antagonist (e.g., Antibody 7, described in Table 1) or subjects known torespond poorly to treatment with a BMP6 antagonist (e.g., Antibody 7,described in Table 1), as the case may be. A control level of expressionmay be derived from biological samples from reference subjects (i.e.,anemia patients known to respond well to treatment with a BMP6antagonist (e.g., Antibody 7, described in Table 1) or subjects known torespond poorly to treatment with a BMP6 antagonist (e.g., Antibody 7,described in Table 1)), or may simply be a numerical standard (e.g.,mean, median, range, [+/−standard deviation]) previously derived fromreference subjects. In some embodiments the control is a reference levelof ferritin expression, ferritin protein, or ferritin activity derivedfrom a subject known to respond poorly to treatment with a BMP6antagonist and the level of ferritin expression, ferritin protein, orferritin activity (as the case may be) from the patient is compared tothis control. In other embodiments, the control is a reference level offerritin expression, ferritin protein, or ferritin activity derived froma subject known to respond well to treatment with a BMP6 antagonist andthe level of ferritin expression, ferritin protein, or ferritin activityfrom the patient to be treated is compared to this control, wherein asimilar (e.g., statistically similar) level of ferritin expression,ferritin protein, or ferritin activity in the patient (relative to acontrol) provides an indication that the patient will have an increasedlikelihood of responding to treatment with the BMP6 antagonist Antibody7, described in Table 7).

In performing any of the methods described herein that requiredetermining the presence or level of ferritin expression, ferritinprotein, or ferritin activity, such determination may be made byconsulting a data repository that contains sufficient information on thepatient's composition to determine whether the patient has the markerlevel of marker) of interest. Preferably, the data repository lists themarker and level of marker of interest in the individual. The datarepository could include the individual's patient records, a medicaldata card, a file (e.g., a flat ASCII file) accessible by a computer orother electronic or non-electronic media on which appropriateinformation or genetic data can be stored. As used herein, a medicaldata card is a portable storage device such as a magnetic data card, asmart card, which has an on-board processing unit and which is sold byvendors such as Siemens of Munich Germany, or a flash-memory card. Ifthe data repository is a file accessible by a computer; such files maybe located on various media, including: a server, a client, a hard disk,a CD, a DVD, a personal digital assistant such as a smart phone, PalmPilot, a tape recorder, a zip disk, the computer's internal ROM(read-only-memory) or the internet or worldwide web. Other media for thestorage of files accessible by a computer will be obvious to one skilledin the art.

Typically, once levels of ferritin expression, ferritin protein, orferritin activity is determined, physicians or genetic counselors orpatients or other researchers may be informed of the result.Specifically the result can be cast in a transmittable form ofinformation that can be communicated or transmitted to other researchersor physicians or genetic counselors or patients. Such a form can varyand can be tangible or intangible. The result in the individual testedcan be embodied in descriptive statements, diagrams, photographs,charts, images or any other visual forms. For example, images of gelelectrophoresis or capture assays can be used in explaining the results.Statements regarding levels of ferritin expression, ferritin protein, orferritin activity are also useful in indicating the testing results.These statements and visual forms can be recorded on a tangible mediasuch as papers, computer readable media such as floppy disks, compactdisks, etc., or on an intangible media, e.g., an electronic media in theform of email or website on internet or intranet. In addition, theresult can also be recorded in a sound form and transmitted through anysuitable media, e.g., analog or digital cable lines, fiber optic cables,etc., via telephone, facsimile, wireless mobile phone, internet phoneand the like. All such forms (tangible and intangible) would constitutea “transmittable form of information”. Thus, the information and data ona test result can be produced anywhere in the world and transmitted to adifferent location. The test result in a transmittable form thus can beimported into the U.S. Accordingly, the present disclosure alsoencompasses a method for producing a transmittable form of informationcontaining levels of ferritin expression, ferritin protein, or ferritinactivity in an individual. This form of information is useful forpredicting the responsiveness of a patient having a disease describedherein, e.g., anemia, with a BMP6 antagonist, for selecting a course oftreatment based upon that information, and for selectively treating apatient based upon that information.

Disclosed herein are methods of predicting the likelihood that a patientwith levels of ferritin protein will respond (e.g., respond withenhanced efficacy, e.g., relative to a general population of patientssuffering from the same or similar disease) to treatment with a BMP6antagonist, comprising detecting the level of ferritin in a biologicalsample from the patient, wherein a level of ferritin described herein,e.g., 1500 ng/mL, is indicative of an increased likelihood that thepatient will respond to treatment with the BMP6 antagonist.

In some embodiments, the method further comprises the step of obtainingthe biological sample from the patient, wherein the step of obtaining isperformed prior to the step of assaying.

In some embodiments, the biological sample is selected from the groupconsisting of synovial fluid, blood, serum, feces, plasma, urine, tear,saliva, cerebrospinal fluid, a leukocyte sample and a tissue sample. Insome embodiments, the biological sample is blood or serum.

In some embodiments, the presence and/or level of ferritin expression,ferritin protein, or ferritin activity is detected by a techniqueselected from the group consisting of immunoassays,immunohistochemistry, ELISA, flow cytometry, Western blot, HPLC, andmass spectrometry.

In some embodiments of the disclosed methods and uses, the BMP6antagonist is a BMP6 binding molecule or a BMP6 receptor bindingmolecule. In some embodiments, the BMP6 binding molecule or BMP6receptor binding molecule is a BMP6 binding molecule. In someembodiments, the BMP6 binding molecule is a BMP6 antibody orantigen-binding portion thereof.

In some embodiments of the disclosed methods and uses, the BMP6 antibodyis Antibody 7, as described in Table 1.

Methods of Treatment and Uses of BMP6 Antagonists

The disclosed methods allow clinicians to provide a personalized therapyfor patients suffering from a disease described herein, e.g., anemiapatients, i.e., they allow determination of whether to selectively treatthe patient with a BMP6 antagonist (e.g., Antibody 7, e.g., as describedin Table 1). In this way, a clinician can maximize the benefit andminimize the risk of BMP6 antagonism in the entire population ofpatients afflicted with a disease described herein, e.g., anemia. Itwill be understood that BMP6 antagonists, e.g., BMP6 binding molecules(e.g., BMP6 antibody or antigen-binding portion thereof, e.g., Antibody7, e.g., as described in Table 1) or BMP6 receptor binding molecules(e.g., BMP6 receptor antibody or antigen-binding portion thereof) areuseful for the treatment, prevention, or amelioration of a diseasedescribed herein, e.g., anemia (e.g., signs and symptoms, etc.) asdisclosed herein, particularly in patients that have a level of ferritindescribed herein.

EXAMPLES

The following examples are provided to further illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Example 1 In Vitro and In Vivo Activity, and PK/PD of Anti-BMP6Antibodies Materials

Test compounds were Antibodies 5, 6 and 7 (Table 8), at a concentrationof ˜8 mg/ml in 50 mM citrate buffer, pH 7.0, 150 mM NaCl and diluted inPBS before animal administration. Male C56BL/6 mice or Sprague Dawleyrats were used (Table 9).

TABLE 8 Properties of BMP6 antagonist antibodies IC50(ug/ml) BMP6Antibody ID Framework KD(nM) BMP6 reporter ANTIBODY 5 VH3_15, Vl1 0.10.06 ANTIBODY 6 VH3_15, Vl1 <0.1 0.08 ANTIBODY 7 VH3_15, Vl1 0.1 0.07

TABLE 9 Animal characteristics Species Strain Category Vendor Gender AgeMouse C57BL/6 wild-type Jackson Male 8-9 (Mus Laboratory, Bar Weeksmusculus Harbor, ME Rat Sprague wild-type Charles River Male 8-12(Rattus Dawley Laboratory, weeks norvegicu Wilmington, MA

For BMP reporter gene assays, a lentiviral vector was constructedcontaining BMP responsive element BRE in the promoter [Korchynskyi etal, 2002. J. Biol. Chem. 277: 4882-91] driving firefly luciferasederived from pGL4-BRE2-Luc2. The lentiviral vector was used to stablytransfect HEP3B hepatoma cell line. The cell line was maintained in EMEMwith 10% fetal bovine serum, 1% Penicillin/streptomycin, and 5 ug/mlBlasticidin. Recombinant human BMP proteins were purchased from R&DSystems.

Brucella abortus Ring Test Antigen (strain 1119-3) in 60 ml bottles werepurchased from U.S. Department of Agriculture, Animal and Plant HealthInspection Service, National Veterinary Services Laboratories, Ames,Iowa. Brucellosis ring test antigen contains a suspension of killed,stained B. abortus strain 1119-3 cells in phenolized buffer. Theconcentration of each 60 mL bottle is approximately 10⁹ particles/ml. A5×10⁹ stock is washed and prepared in the following manner. First, 60 mlbottles are removed from refrigerator and mix completely. 500 ml of BAis then transferred into 500 mL centrifuge bottle. These are thencentrifuges at 10,000 rpm for 15 minutes using an ultracentrifuge. Thesupernatant is removed and re-suspend in 100 mL PBS, resulting a 5×10 ⁹particles/ml stock, which was aliquoted and frozen at −80° C.

Animal Maintenance Conditions

Animals were socially housed in micro-isolator solid-bottom cages duringthe acclimation and study periods. Animals were kept under a standardlight cycle as follows: 12 hours dark, 12 hours light (lights on: 6:30AM, lights off: 6:30 PM) with room temperature 21-23° C. and humidity30-70%. During the acclimation and study periods, animals were givenaccess to rodent diets and water ad libitum (ad lib).

Experimental Conditions Determination of the Antibody Activity in BMPReporter Gene Assay

In atypical assay, 0.6×10⁴ BRE-Luc2 HEP3B cells were seeded on 384-wellplates in 25 ul of basic culture medium except that serum was reduced to2%. On the next day antibodies diluted in PBS were added, following bythe addition of BMP6 to a final concentration of 10 ng/ml. The volumewas brought to 50 ul with EMEM media without any serum, making finalserum concentration 1%. As counter assays, activation with BMP2/4/7 wasdone in parallel. BrighGlo assay (Promega) was performed 24 hourspost-antibody addition according to manufacturer's instruction, using anEnvision plate reader (PerkinElmer). Data were calculated as percent ofinhibition for each antibody compared to full reporter activation by acontrol antibody.

Single Dose Antibody Pharmacokinetics Study in Rat

The rat PK triaging study is not intended to determine classical PKparameters with a defined statistical certainty, but rather to providean estimate of the serum half-life for the test antibody. 3 animals wereinjected with a single IV dose of the antibody.

For mouse dose-response PK/PD study, animals were divided into in 2separate cohorts of equal numbers. Each cohort includes both vehicle-and compound-treated mice. One cohort was subjected for analyses on day2, 4 whereas the second cohort was analyzed on day 6, 8 after antibodyinjection. The reason for the separation of cohorts is to reduce theneed for serial bleeding so that the impact on serum iron parameters iskept minimal. The animal groups are shown in Table 10.

TABLE 10 Design, animal allocation and test article doses Dose (mg/kg,Experiment Group Number IV) Frequency Mouse PK/PD Control hIgG 5 0.5Once 0.05 mpk 5 0.05 Once ANTIBODY 6 0.1 mpk 5 0.1 Once ANTIBODY 6 0.5mpk 5 0.5 Once ANTIBODY 6 Mouse BA Sham (No BA) 6 0 0 anemia BA, EPO + 62 Once control hIgG BA, EPO + 6 2 Once ANTIBODY 5 BA, EPO + 7 2 OnceANTIBODY 6 BA, EPO + 6 2 Once ANTIBODY 7

Establishment of Anemia of Inflammation in Mice and TherapeuticTreatment

5×10⁸ BA particles for injection are prepared in the following manner(example for 10 mice). Starting concentration needs to be 2.5×10⁹particles/ml since 200 μl/mouse will be injected. Dilute stock 2-foldusing PBS. For example, 10 mice times 0.200 μl=2 ml+20% overage=2.2 mLof 2.5×10⁹ particles ml needed. 1.1 ml BA stock+1.1 mL PBS. BAadministration 1 to 8 days before ESA treatment was shown to result in ablunted HGB response 6 to 7 days later.

C57BL/6 mice were injected with BA (3×10⁸ particles/mouse) and serum IL6levels were measured 5 hours later by ELISA (KMC0061, Life Technologies)to determine the inflammatory response. Animals with a IL6 concentrationlower than the 95% confidence interval of the mean for all BA-treatedanimals were excluded from the study, resulting in fewer than 5-6 micein some groups. This exclusion process was carried out to lessen thepossibility of false-positive results produced by including animals thatdid not have sufficient inflammation to blunt ESA response. After theexclusion process, mice were injected IV with the antibodies asindicated on day 6, and EPO (100 g/kg subcutaneous darbepoetin alfa,Amgen) was administered at 100 mg/kg on day 7 relative to BA treatment.Response to ESA and antibody therapy was measured 6 days later.

Analyses of Pharmacokinetics, Pharmacodynamics, and Efficacy Endpoints

For mouse and rat PK/PD studies, serum samples were collected atindicated time points post antibody injection. Aliquots of the sera wereused to determine circulating antibody concentration through automatedhigh-throughput immunoassay system (Gyros) with biotinylated anti-humanIgG as primary capture antibody. A second serum aliquot of each samplewas used for quantitative colorimetric iron assay (Quntichrom, DIFE-250,Bioassay Systems). A third aliquot was processed for LC-MS quantitationof the rat or mouse hepcidin-25 peptide, following a modified proceduredescribed earlier. Li et al. 2009. J. Pharm. Tox. Meth. 59: 171-80.

For BA-induced anemia and antibody treatment study, a final bleed inEDTA-coated BD Microtainer tubes were obtained at termination throughcardiac puncture. The whole blood was used for Complete Blood Countanalyses on an XT-2000iV hematology analyzer. Efficacy endpoints includeHGB, HCT RETA, and RET-HE.

Statistical Analyses

One-way analysis of variance (ANOVA) followed by Bonferroni's post hoctest was carried out to analyze group differences (with p<0.05considered significant) in hematology parameters. Data are reported asmeans±SEM.

Results Biological Activity of BMP6 Antagonist Antibodies in CellularBMP-Dependent Transcriptional Assays

All three BMP6 antagonist antibodies 5, 6 and 7 fully inhibit thebioactivity of recombinant human BMP6-induced BMP reporter (BRE-luc)activity in human hepatoma cell line Hep3B (IC50=0.4 nM against 0.3 nMrhBMP6) and therefore is active at a 1:1 Ag/mAb molar ratio or better.The antibodies demonstrated good selectivity over the related BMP familyproteins including BMP2, 5, and 7, with a window of 500 fold or more.See FIG. 1.

Snapshot Pharmacokinetics and Pharmacodynamics Profiles of BMP6Antagonist Antibodies in Rat

Single dose triage pharmacokinetics study in Sprague Dawley rats wasperformed for BMP6 antibodies 5, 6 and 7, through IV injection viajugular vein catheter at 10 mg/kg body weight. Comparing the totalantibody concentration-time relationship (particularly t_(1/2), MRT) inserum of the three antibodies with a standard profile suggestedcharacteristics consistent with atypical human IgG (see FIG. 2 and Table11). There is no evidence of target-mediated drug disposition. At thisdose, all BMP6 antibodies suppressed serum hepcidin to below detectionlevels by day 1 post injection. The sustained strong suppression ofhepcidin expression was still evident by day 16, suggesting a longduration of activity. Correspondingly, a transient peak rise incirculating iron concentration was observed on day 2 after antibodyinjection and the levels remain elevated by day 16.

Serum antibody concentration was measured overtime after a singleantibody injection. Samples were collected at 1 hr, 6 hr, 1, 2, 4, 8,16, 28 days post dose (10 mg/kg, IV).

TABLE 11 Key parameters in single dose rat triage PK study ParametersANTIBODY 5 ANTIBODY 6 ANTIBODY 7 T_(1/2) (days) 9.1 7.8 9.2 C_(max)(ug/ml) 140.7 189.0 146.2 Mean resident 8.6 7.0 6.9 time (days)

As well, total serum concentration of Antibody 7 (both free andBMP6-bound) was measured in rats and cynomolgus monkeys following asingle IV injection of Antibody 7 (in rats, at doses of 10, 3, 1, 0.3,0.1 and 0.03 mg/kg; in monkey at 3 mg/kg) at the indicated times byELISA with an LLOQ of 46 ng/mL (dotted line) rats and a LLOQ of 0.2ug/mL (dotted line) in monkey. The results are shown in FIGS. 9 (rat)and 12 (monkey).

Dose-Dependent Response in Serum Iron Parameters After BMP6 AntibodyTreatment in Mice and Cynomolgus Monkey

To further define dose-dependent response of iron metabolism to BMP6antibody treatment, naive C57BL/6 mice were injected with increasingdose of Antibody 6, ranging from 0.02 to 0.5 mg/kg, as indicated.Antibody 6 was chosen as representative of the 3 antibodies since theyshare similar framework, rodent PK profile and in vitro activities. Asingle dose of 0.5 or 0.1 mg/kg significantly suppressed serum hepcidinand accordingly increased serum iron concentration 2 days aftertreatment. However, only at 0.5 mg/kg, was a strong sustained effect oniron metabolism observed up to 8 days post injection. See FIG. 3. Theseresults suggest dose-dependent, saturable target neutralization can bereadily achieved using potent BMP6 antagonist antibodies.

See FIG. 3, Dose-dependent effects of a BMP6 antibody on serumbiomarkers of iron metabolism Top: Serum hIgG concentration over timefollowing a single IV injection of Antibody 6 at the indicated doses.Bottom: Left panel is quantitative analysis of serum hepcidinconcentration after a single Antibody 6 or control human IgG injection,whereas right panel is serum iron concentration.

Similar experiments were performed with Antibody 7. Dose- andtime-dependent suppression of circulating serum hepcidin by Antibody 7was tested in male Sprague-Dawley rats. Serum samples were collected at0.25, 1, 2, 6 hr, and 1, 2, 4, 7 and 14 d post-dose after a single doseof Antibody 7 was administered by IV injection at a dose ranging from0.03 mg/kg to 10 mg/kg. Serum hepcidin levels were measured by LC/MSwith a LLOQ 9 ng/mL. In the same animals, serum iron levels were alsomeasured. The results are reported in FIG. 11.

These results indicate that the anti-BMP6 antibodies of the presentinvention are able to cause a dose-dependent increase in serum iron. Theeffects were robust and persisted for at least 2 weeks after antibodyadministration.

The effects on serum iron parameters in response to anti-BMP6 antibodywas also tested in cynomeolgus monkey. Male Cynomogus monkeys were givena single intravenous injection of Antibody 7 at a dose of 3 mg/kg. Atindicated days post injection, serum samples were collected and analyzedfor total serum iron (Fe) and hepcidin concentration. The results areshown in FIG. 13. Data from 3 individual animals are presented (plottedagainst the pre-dose baseline levels). Mean values are indicated by the“x” line. An increase in serum iron and suppression of serum hepcidinwere observed 24 hr after antibody administration and the effectsremained (relative to pre-dose levels) through the end of the 28-daystudy. These results indicate that the BMP6 antibodies of the inventionpotently induce hepcidin expression and reduce circulating ironconcentration in non-human primates.

Effect of BMP6 Antibodies on Red Cell Parameters in Inflammation-Driven,ESA-Resistant Anemia in Mice

Experiments were performed to evaluate the therapeutic utility of theanti-BMP6 antibodies in a mouse model of anemia of inflammation. SeeFIG. 4. Mice treated with abortus antigen (BA) developed anemia 6 dayslater. Anemic animals were treated with anti-BMP6 plus antibodyrecombinant erythropoietin (EPO) initiated at one day apart, and theeffect of antibody therapy on anemia progression was monitored at day 13relative to BA. HGB and HCT values decreased between onset of treatmentand day 13, which was resistant to EPO treatment alone. Combined BMP6antibody and EPO treatment effectively restored EPO response andsignificant raised HGB and HCT levels. This effect was associated with aconcomitant stimulation of erythropoietin activity, as reflected bypersistent increase in RETA, as well as restored reticulocyte hemoglobincontent, suggesting a correction of heme synthesis due to functionaliron deficiency in the erythropoiesis compartment.

See FIG. 4, therapeutic treatment of BMP6 Antibody in an ESA-resistantanemia of inflammation mouse model. Top: Experimental scheme ofBA-induced ESA-resistant anemia of inflammation model. Bottom:Erythropoiesis parameters at 13 days after BA treatment. HGB:hemoglobin; HCT: hematocrit; RETA: reticulocyte count; RET-HE:Reticulocyte hemoglobin equivalent.

-   *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus BA+EPO+hIgG1

Example 2 Clinical Plan for Testing of BMP6 Antibodies in Humans

Clinical Trial Plan: Assessment of therapy using antibodies orantigen-binding fragments thereof that bind human BMP6.

Patients with end-stage renal disease (ESRD) produce little, if anyerythropoietin (EPO) and generally require periodic administration ofexogenous EPO and intravenous (IV) infusions of iron to enableEPO-induced synthesis of Hgb. Up to one third of chronic hemodialysis(HD) patients do not respond adequately to EPO, owing primarily tointracellular sequestration of iron. Hepcidin is primarily cleared bythe kidney, but removal by dialysis is insufficient. Therefore, chronicHD patients tend to have significantly elevated hepcidin levels, whichblock mobilization of iron for erythropoiesis. IV iron therapy is nolonger effective or recommended once body iron stores reach a criticallevel (indicated by high serum ferritin levels). Current guidelinesrecommend against giving IV iron to anemic dialysis patients with highferritin levels, and these patients may therefore receive even higherEPO doses, with the potential associated risk of EPO hypo-responsiveanemia (Kidney Disease Improving Global Outcomes (KDIGO) Anemia WorkGroup 2012). EPO hypo-responsive anemia imparts a significantlyincreased risk of all-cause mortality related to both anemia and higherEPO dose in hemodialysis (Kilpatrick et al 2008, Lopez-Gomez et al 2008,Fukuma et al 2012) and peritoneal dialysis patients (Suttorp et al2013). The isolated antibodies or antigen-binding fragments thereof thatbind human BMP6 of the present disclosure may benefit chronic kidneydisease patients with iron-restricted anemia by improving hemoglobin(Hgb) levels while simultaneously reducing EPO and IV iron dosing needs.A lower EPO resistance index (ratio of EPO dose vs. Hgb level) iscorrelated with a lower mortality risk.

In summary, the goal of therapy using the isolated antibodies orantigen-binding fragments thereof that bind human BMP6 of the presentdisclosure is to mobilize sequestered iron, which may then reduce EPOand iron dose needs and improve Hgb levels, all of which is expected toimprove patient outcomes. This is a first-in-human, single dose study oftherapy using isolated antibodies or antigen-binding fragments thereofthat bind human BMP6. This study will assess safety, tolerability,pharmacokinetics, pharmacodynamics and efficacy in a chronichemodialysis patient population. The purpose of this study is toevaluate whether therapy using isolated antibodies or antigen-bindingfragments thereof that bind human BMP6 warrants further clinicaldevelopment in anemia associated with chronic kidney disease.

Investigational Plan Study Design

This is a first-in-human, two-part, single-dose, non-confirmatory studyof an isolated antibody or antigen-binding fragment thereof that bindshuman BMP6 to assess safety, tolerability, PK, PD and efficacy in achronic hemodialysis patient population. Part 1 is a first-in-human,single-dose, open-label dose-finding study. Part 2 is a randomized,double-blind, placebo-controlled, single-dose study that will comparetwo dose levels of an isolated antibody or antigen-binding fragmentthereof that binds human BMP6.

Safety assessments will include physical examinations, ECGs, vitalsigns, standard clinical laboratory evaluations (hematology, bloodchemistry, serum iron indices) adverse event and serious adverse eventmonitoring.

Part 1

The aims of Part 1 are (a) to evaluate single-dose safety, PK, PD, andtolerability, and (b) to determine the minimum PAD of an isolatedantibody or antigen-binding fragment thereof that binds human BMP6,defined as the lowest dose tested in Part 1 that results in an increasein Hgb (median change from baseline ˜0.5 g/dL) at 29 days post-dose.

During Part 1, a screening visit will take place, where the patient'seligibility to enter the study will be determined (FIG. 6). Eligiblepatients will be admitted to the study site and re-evaluated foreligibility criteria during the baseline visit. All baseline safetyevaluation results must be available and reviewed prior to dosing.

FIG. 6 provides and overview of the study design for Part 1. Patientswill be asked to arrive at the study site on Day 1, directly followingtheir routine dialysis visit. Patients will then receive an infusion ofan antibody or antigen-binding fragment thereof that binds human BMP6(exact dose will be dependent on cohort). If possible, dosing shouldpreferably take place on a dialysis day prior to two inter-dialysis days(e.g. Friday or Saturday), and will occur following that day's dialysissession. However, if not possible, then dosing may occur on a dialysisday not preceding two inter-dialysis days. Following dosing, the firsttwo patients in Part 1 will be domiciled for at least 48 hours forsafety and PK/PD assessments. Patients will return to the study site atDays 4 and 6 for PK/PD assessments, and then weekly for a total of 29days for PK assessments, and a total of 12 weeks for safety assessment,with an end-of-study visit at approximately Day 85. Study visits,including all laboratory tests other than post-dialysis PK assessments,should take place before the patients scheduled dialysis visit.

Part 1 will be initiated with a dose that is predicted to be notpharmacologically active. The dose will be adjusted for each subsequentPart 1 cohort, based on each cohorts median change in Hgb following asingle dose of an isolated antibody or antigen-binding fragment thereofthat binds human BMP6 and transferrin saturation (TSAT) level asdiscussed in the Statistical Considerations section and shown in FIG. 7.The aim of this decision tree is to identify the minimum feasible dosethat induces iron mobilization (as indicated by transferrin saturation(TSAT) levels >50% observed in at least 4 patients in the 6-patientcohort at one week post-dose) and increases Hgb. If iron is mobilizedbut Hgb does not increase by at least 0.5 g/dL at 29 days post-dose,then the clinical data will be analyzed to assess potential confoundingfactors (e.g. blood loss due to excessive non-study phlebotomy). Theapplicable Investigators and representative(s) from the Sponsor willreview each cohort's adverse events and will assess these events in thecontext of (a) known medical issues associated with chronic renalfailure and (b) an nonclinical toxicology findings. Subsequent cohortswill not be dosed until the Investigators and Sponsor indicate that itis safe to proceed.

FIG. 7 provides the algorithm for adjustment of doses in Part 1. Bloodwork including Hgb measurements will occur pre-dialysis. The startingdose will be 0.01 mg/kg. In Part 1, patients will be assigned to one ofup to 6 open label dose cohorts of up to 6 patients each. The minimumPAD of an isolated antibody or antigen-binding fragment thereof thatbinds human BMP6, as defined above, will be the lower dose arm selectedfor Part 2. The dose for each subsequent cohort may be adjusted higheror lower, as shown in FIG. 7. If the lowest feasible dose (0.001 mg/kg)results in a median increase in Hgb of ≥0.5 g/dL, it will be the minimumPAD and the lower dose selected for Part 2 and the next highest doseevaluated in Part 1 will be the higher dose arm for Part 2. If thehighest dose (0.1 mg/kg) evaluated in Part 1 is the minimum PAD, Part 2will proceed with 2 arms only: placebo and minimum PAD. In the eventthat additional dose cohorts are needed in Part 1, these cohorts will beadded as described in herein.

Each Part 1 cohort will include 6 patients. The first 2 patients in thefirst cohort of Part 1 will be dosed at least 7 days apart. Timing ofsubsequent Part 1 cohort patient doses will occur as is feasible for therespective site's schedule and support resources. All Part 1 patientswill be followed for 12 weeks following the dose.

Part 2

The aims of Part 2 are (a) to evaluate safety, PK, PD, and tolerabilityand (b) to determine efficacy based on Hgb changes in response to singledose of an antibody that binds human BMP6 vs. placebo. Part 2 willinclude up to three arms: Up to two Ab dose arms and a placebo arm (FIG.8). The two Ab dose arms will be derived from data generated in Part 1.Part 2 will include approximately 60 patients with a randomization of1:1:1 to the three arms. If, in Part 1, the minimum PAD is also thehighest dose (0.1 mg/kg) evaluated, then Part 2 will have only two arms:minimum PAD and placebo. In this case, 40 patients will be randomized tothe two arms with a randomization ratio of 1:1. Sample size of Part 2may be adjusted based on the variability of the change from baseline inHgb in Part 1.

FIG. 8 provides a study design for Part 2. During Part 2, a screeningvisit will take place, where patient's eligibility to enter the studywill be determined. Eligible patients will be re-evaluated as pereligibility criteria during the baseline visit. All baseline safetyevaluation results must be available and reviewed prior to dosing.

Patients will be asked to arrive at the study site on Day 1, directlyfollowing, their routine dialysis visit. Patients will then receiveeither an infusion of Ab or placebo, as determined by randomizationassignment. If possible, dosing should preferably take place on adialysis day prior to two inter-dialysis days (e.g. Friday or Saturday),and will occur following that day's dialysis session. However, if notpossible, then dosing may occur on a dialysis day not preceeding twointer-dialysis days. Patients will return to the study site on Days 4and 6, then weekly for follow up assessments. During follow up visitspatients will undergo routine safety assessments and PK data will alsobe collected 85 days. Study visits may take place following thepatient's scheduled dialysis visit, in order to fit with the dialysisschedule of the patient. All patients enrolled in Part 2 will befollowed for 12 weeks following the dose of Ab (or placebo).

EPO Dose Management (Both Parts)

Individual EPO dose adjustments during both Parts will be managed as pereach dialysis site's standard of care protocol. Site protocols will bereviewed as part of site assessment, and will be checked for compliancewith standard of care guidelines (KDIGO Clinical Practice Guideline forAnemia in Chronic Kidney Disease Anemia Work Group 2012). Patients whoachieve a Hgb level of ≥13 g/dL at any time during the study may bemanaged with therapeutic phlebotomy, at the discretion of theinvestigator, in addition to site-specific guidelines for managing Hgbvalues above target levels.

Intravenous Iron Management (Both Parts)

Patients receiving loading doses of IV iron (100 mg/week) will beexcluded from the study. Patients receiving weekly maintenance IV iron(<100 mg/week) may be included in this study. The weekly maintenance IViron dose will be held at the beginning of week 1 of Ab dosing. Ironindices will be monitored during the first week post-Ab dosing, andrescue iron therapy and maintenance IV iron management will followstandard of care guidelines as per the managing hemodialysis unit'sprotocol. Site protocols will be reviewed as part of site assessment,and will be checked for compliance with standard of care guidelines(KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney DiseaseAnemia Work Group 2012).

Rationale of Study Design Rationale for Two-Part Study Design

The rationale for two parts in the same patient population is toidentify the minimum PAD safely and efficiently, aiming to minimize thenumber of patients and cohorts exposed to potentially sub-therapeuticdoses. Part 2 will assess the efficacy of the minimum PAD, and one doselevel above the minimum PAD (as determined in Part 1), in comparisonwith a placebo group.

Part 1 is designed to evaluate single-dose safety, tolerability, PK/PD,as well as the minimum PAD of Ab in an open label study. The minimum PADwill be determined based on each dose cohort's median change in Hgb at29 days following Ab dosing. The rationale for the PAD determinationcriteria is that clinically meaningful responses to EPO may require upto 4 weeks following an EPO dose change. If Ab mobilizes iron in thetarget population, then that may enable the patient's current EPO doseto exert a more robust erythropoietic effect. The 29 days Hgb rangeslisted in the PAD determination criteria are based on clinicallysignificant & safe rates of increase in Hgb in response to an EPO dose(˜0.5 g/dL over 29 days). The rationale for seeking the minimum PADrather than a maximal effect is that an overly robust Hgb response is asafety risk in this patient population, as reflected by the target Hgbranges in the current standard of care guidelines (Kidney DiseaseImproving Global Outcomes (KDIGO) Anemia Work Group 2012). The goals ofthe safety and tolerability assessments in Part 1 are (a) to identifysafety signals, and (b) to inform dose adjustment decisions, ensuringthat the doses selected for Part 2 (minimum PAD+1 dose higher) aresuitable for further evaluation of both safety and efficacy relative toplacebo. While Part 1 is inadequately powered to afford an unbiasedassessment of safety, the placebo group and larger sample sizes in Part2 will enable an unbiased safety assessment at the minimum PAD and onedose higher. In addition to safety, tolerability, and PK/PD, Part 2 isdesigned to assess efficacy vs. placebo in a double-blind study.Efficacy assessment will be based primarily on Hgb, with EPO resistanceindex (ERI=weekly weight-adjusted EPO dose divided by Hgb) as a keysecondary endpoint. ERI provides a quantitative measure of the amount ofEPO needed to achieve a given Hgb value, and therefore providesclinically important information in addition to Hgb alone.

Rationale for FIH in Dialysis Patients

This first-in-human (FIH) study will be conducted in chronichemodialysis (HD) patients rather than healthy volunteers (HV).Evaluation of safety, tolerability, and PK/PD response to anti-humanBMP6 Ab in HV is likely not translatable to chronic HD patients forseveral reasons:

Unlike HV, chronic HD patients with anemia, high serum ferritin, and lowTSAT have chronically accumulated intracellular iron stores. Therefore,safety, tolerability, and pharmacological effects related to ironmobilization in response to low doses of Ab are most appropriatelyevaluated in chronic HD patients. In HV with normal renal function,hepcidin (of which BMP6 is a key regulator) is filtered by the kidneyand is excreted efficiently in the urine, leading to low circulatinglevels. In contrast, hepcidin is filtered less efficiently andtransiently by dialysis, leading to higher circulating levels in chronicHD patients (Zaritsky et al 2010). Furthermore, normal kidneys willadjust endogenous EPO levels dynamically and a change in Hgb may not beevident in response to Ab. Therefore, safety and tolerability related tomodulation of hepcidin by the BMP6 pathway and the effect of Ab on Hgbare most appropriately evaluated in chronic HD patients.

Rationale for Target Patient Population

This study is designed to evaluate an anti-human BMP6 Ab in the settingof EPO-hypo-responsive, iron-restricted anemia. Established clinicalguidelines (KDIGO Clinical Practice Guideline for Anemia in ChronicKidney Disease Anemia Work Group 2012) define EPO hypo-responsiveness asthe need for two increases in EPO dose, up to 50% above the stable dose,to maintain a stable Hgb concentration. The proposed eligibilitycriteria are designed to select for stable chronic HD patients withanemia, and clinical indicators of iron restriction: increased ferritinand low TSAT (TSAT=serum iron/total iron binding capacity; TSATcorrelates very closely with serum iron). Furthermore, adjustments inEPO and IV iron doses will adhere to strict standard of care targets forHgb, TSAT, and ferritin. This design reduces the risk of over-shootingdesired Hgb targets because changes in iron and hematologic parameterswill continue to be managed as per standard of care. Furthermore,patients receiving loading doses of IV iron within 1 week prior tobaseline will be excluded. Patients receiving maintenance IV iron may beincluded (if all other eligibility criteria are met). The rationale forincluding these patients is that current standard of care in the USAdictates that Hgb and TSAT be maintained within narrow limits, andtherefore, full withdrawal of maintenance iron therapy for the purposeof meeting lower TSAT eligibility criteria would place patients at riskfor TSAT below 25%, necessitating a course of IV iron loading doses asper standard of care. However, eligible patients on maintenance IV ironwill have their weekly IV iron dose held at the beginning of the week ofAb dosing, and will resume maintenance IV iron therapy only asdetermined by site's standard of care protocol, based on monitored ironindices.

Rationale of Dose/Regimen, Duration of Treatment Starting Dose Rationale

The maximum recommended starting dose (MRSD) was calculated based on theno adverse effect level (NOAEL) from the 13-week (14-dose) GLPtoxicology studies conducted in rats and cynomolgus monkeys. Animalsreceived weekly IV bolus doses of 0.1, 1, 10, and 100 mg/kg. The 1 and100 mg/kg dose groups (only) were subsequently followed for 16 weeks inrats or 24 weeks in cynomolgus monkeys after the last dose of anisolated antibody or antigen-binding fragment thereof that binds humanBMP6. The MRSD was estimated by first calculating the human equivalentdose (HED) for the NOAEL from these studies (0.1 mg/kg)—an approachdeemed appropriate for drugs with a molecular weight >100 kDa—andsubsequently applying a safety factor of 10 to account for differencesbetween nonclinical species and patients, such as the amount of storediron and the demand for erythropoiesis. PK parameters for thenonclinical species were inferred from the toxicokinetic (TK) datacollected during the IND-enabling toxicology studies. Corresponding PKparameters in patients were then estimated using allometric scaling, andthese parameters were used to predict free an isolated antibody orantigen-binding fragment thereof that binds human BMP6 concentration asa function of time in patients for a given dose. Comparing the TK datafrom the toxicology studies to the model-based an Ab PK in patientsindicated that a dose 10-fold lower than the NOAEL/HED was predicted toyield a (minimum) 10-fold margin based on Ab concentration.

The maximal levels of serum iron observed in response to Ab in animalstudies may underestimate the predicted human iron response to Ab,because HD patients who have been administered IV iron therapy likelyhave higher tissue stores of iron than healthy animals. However, unlikehealthy, non-anemic animals, HD patients are expected to utilize thereleased serum iron for erythropoiesis; therefore animal models mayoverestimate the duration of iron elevation. The liver pathologyobserved in the 13-week studies was not observed in the 4-week studies,suggesting that the toxicities owe to the cumulative exposure to serumiron rather than a response to the acute release of iron.

To account for the anticipated differences in stored iron betweennonclinical species and patients, MRSD was also predicted based on amodel-based analysis of serum iron concentrations. The cumulativeexposure to serum iron that resulted in the toxicology findings wasrepresented as an iron area-under-the-curve (Fe AUC). In this approach,the Fe AUC calculated for the NOAEL dose (0.1 mg/kg) was regarded asbeing adequately safe. The Fe AUC at the proposed MRSD in patients wasthen predicted and compared to the Fe AUC in nonclinical species at theNOAEL. Because the model-predicted serum iron exposure at the MRSD inpatients was >10-fold less than that at the NOAEL in nonclinicalspecies, decreasing the NOAEL/HED by a safety factor of10 was deemedadequate for the estimation of a MRSD. The proposed MRSD is therefore0.01 mg/kg.

Dose Adjustment Rationale

For this study, the maximum test dose (xmax) will be the HEDcorresponding to the NOAEL in each of the 2 IND-enabling toxicologystudies: 0.1 mg/kg. The minimum feasible test dose (xmin) is the lowesttechnically feasible dose based on compatibility studies: 0.001 mg/kg.The MRSD (x0) will be evaluated according to the safety, TSAT, and Hgbcriteria (FIG. 7). If x0 results in a median change in Hgb <0.5 g/dLrelative to pre-dose, selection of x+(FIG. 7) will be guided by linearlyextrapolating on a natural base logarithmic scale (in anticipation of asigmoidal dose-response relationship) between x0 and xmax. Provisionaldoses for this dose escalation are provided above. These provisionaldoses may be adjusted based on the review of data during the informalinterim analysis between each cohort. This approach will continue untileither the minimum PAD is identified or xmax is reached. If max resultsin a median change in Hgb <0.5 g/dL, the safety of this dose will beevaluated and a decision will be made whether to amend the protocol toadd additional cohorts at doses that exceed the max, based on safety,PK, and PD data. If the highest dose tested in Part 1 results in amedian increase in Hgb <0.5 g/dL, does not increase TSAT above 50%, andthat dose is below xmax, the protocol may be amended to add additionalcohorts.

If x0 instead results in a median change in Hgb ≥0.5 g/dL relative topre-dose, the dose for the next cohort will be adjusted to xmin. If xminalso results in a median change in Hgb ≥0.5 g/dL, xmin will be deemedthe minimum PAD, and xmin and x0 will be evaluated in Part 2. If minresults in a median change in Hgb <0.5 g/dL and TSAT ≤50% (FIG. 7),doses will be increased by linear extrapolation within the interval(xmin, x0) on the natural base logarithmic scale until either theminimum PAD is identified or until 6 doses (cohorts) have beenevaluated. Provisional doses within the interval (xmin, x0) are providedabove. These provisional doses may be adjusted based on the review ofdata during the informal interim analysis between each cohort. Theminimum PAD will be defined as the lowest dose tested that results in amedian change in Hgb ≥0.5 g/dL relative to pre-dose.

The Ab will be administered as a single dose IV infusion to ensure serumiron exposure (Fe AUC) less than that associated with adverse findingsin nonclinical toxicology studies. The Ab solution will be infusedimmediately following the hemodialysis session on Day 1 to minimize thepotential impact of dialysis on PK or immediate post-dose ironbioavailability. The additional approximately 30 minutes of dosinginfusion following dialysis (on dosing day only) is not expected to poseany significant risk or discomfort to patients.

Rationale for Choice of Comparator

Placebo is employed as a comparator in Part 2 to enable unbiasedevaluation of clinical outcomes.

Purpose and Timing of Interim Analyses/Design Adaptations

In Part 1, after each cohort of 6 patients finishes the week 4 post-doseassessment, an informal interim analysis will be conducted to make thedose adjustment decision for the next cohort. Safety and PD markers willbe reviewed by all members of the dose adjustment team, including theapplicable Investigators and representative(s) from the Sponsor. Newcohorts will be triggered only if safety and tolerability is confirmed,and if the PD conditions are met as described in FIG. 7. There will beup to 5 informal interim analyses in Part 1. A formal interim analysisis planned after all patients from the last cohort of Part 1 finish theweek 4 post-dose assessment to evaluate the clinical effects of dosesinvestigated, and potentially trigger additional non-clinical studies,and may inform subsequent clinical studies. Body temperature, bloodpressure, pulse rate, ECG evaluation, blood chemistry, hematology ironindices, EPO resistance index, and adverse events collected through Day29 of the last cohort conducted in Part 1 will be included.

The minimum PAD and a dose one level higher than the minimum PAD will beselected for Part 2. If the lowest possible tested dose induces a Hgbincrease of ≥0.5 g/dL, the two lowest doses tested will be selected forPart 2.

Risks and Benefits

The potential benefit for patients participating in this study mayinclude reduced EPO and IV iron needs, and improved Hgb levels duringthe time of treatment and for some time beyond.

The risk to patients in this trial will be minimized by adherence to theeligibility criteria, and close clinical monitoring of all patients (anddomiciling the first two patients in Part 1) for the first 48 hoursfollowing administration Ab.

The potential risks associated with iron mobilization include (a) ironredistribution to tissues and organs such as the spleen, liver, heart,pancreas, and pituitary, and (b) a small increased susceptibility tobacterial infection, particularly in patients with indwelling vascularcatheters. Several of the eligibility criteria reduce the risk ofcomplications. Increased levels of liver function tests may be seen inassociation with iron redistribution. Liver function will be monitoredin parallel with hematologic and iron parameters. Overshooting ofstandard of care Hgb targets may result in polycythemia. Management ofHgb, EPO therapy, and iron therapy may be undertaken

HD patients who have been administered IV iron therapy may have highertissue stores of iron than healthy animals; therefore the maximal levelsof serum iron observed in patients treated with an isolated antibody orantigen-binding fragment thereof that binds human BMP6 may exceed thoseseen in animal studies. However, unlike healthy animals, HD patients areexpected to utilize the released serum iron for erythropoiesis;therefore animal models may overestimate the duration of iron elevation.The model-predicted exposure to Ab (e.g., Cmax, AUC) at the MRSD isanticipated to be 10-fold less than that observed at the NOAEL innonclinical studies. This exposure is not expected to result in serumiron exposure (AUC) levels associated with the elevated livertransaminases and single cell necrosis in the liver observed inpreclinical studies. Escalation of Ab dose to the NOAEL will occurfollowing; described safety evaluations. Clinical experience withpatients with chronic iron overload as well as those who receiveparenteral iron likely does not necessarily predict the effects that mayoccur from acute increases in intracellular iron induced by Ab.Therefore the potential risk Ab-induced acute iron toxicity is probablylow. Acute iron toxicity may affect the heart, liver, and/or pancreas.Clinical manifestations of acute iron toxicity may include cardiacconduction defects, elevated liver transaminases, and glucoseintolerance/hyperglycemia. Severe acute iron toxicity may also includemetabolic acidosis, electrolyte abnormalities, and neurologicmanifestations. In the event that acute iron toxicity occurs, patientsmay be emergently treated with iron chelation therapy such asdeferoxamine combined with hemodialysis. A maximum of 134 mL (Part 1)and 172 mL (Part 2) of blood is planned to be collected over a period of115 days, from each patient as part of the study. Additional samples formonitoring of any safety findings would be in addition to this. This isnot considered to be a risk for this population.

No reproductive toxicity studies have been performed to date with theanti-human BMP6 antibodes. Potential effects on male or femalereproductive organs have been assessed by careful standardhistopathological examination of the ovaries and testes and accessoryreproductive organs in the 13-week toxicity study in cynomolgus monkeys.No treatment-related effects were observed. BMP6 knock out mice showeddelayed sternum ossification and iron overload (Meynard et al 2009).

Significant fetal and maternal morbidity and mortality is associatedwith chronic hemodialysis. In one retrospective cohort study comparingwomen on chronic hemodialysis (267 births) with women who received arenal transplant (264 births), women on hemodialysis demonstrated higherrates of placental abruption, blood transfusion,small-for-gestational-age babies, fetal deaths, and maternal deaths(Saliem et al 2015). Therefore, women of childbearing potential shoulduse highly effective contraception to prevent pregnancy during anisolated antibody or antigen-binding fragment thereof that binds humanBMP6 administration and for 125 days following the last dose.

Population

The study population will be comprised of patients with end-stage renaldisease who require chronic hemodialysis therapy at least two times perweek, and who have clinical evidence of functional iron-deficiencyanemia, defined as anemia in the presence of apparently sufficient ironstores as determined by ferritin and transferrin saturation levels. Part1 includes a plan to evaluate up to 36 patients initially in 6 cohorts(6 patients/cohort). If after 6 cohorts, no effects on TSAT and Hgb areseen, and there are no safety concerns (as determined by the applicableInvestigators and representative(s) from the Sponsor), up to 2additional 6-patient cohorts may be added (totaling 48 patients in Part1). Part 2 consists of up to 3 arms (2 dose levels selected for furtherevaluation from Part 1, and a placebo group), with up to approximately20 patients per arm (totaling 60 patients in Part 2). Therefore,enrollment of a total of approximately 96 patients (up to a maximum of108) is planned, of which approximately 60 will be randomized in Part 2.Approximately 60 patients (12 in Part 1, 48 in Part 2) are expected tocomplete the study. The investigator must ensure that all patients beingconsidered for the study meet the following eligibility criteria. Noadditional criteria should be applied by the investigator, in order thatthe study population will be representative of all eligible patients.

Patient selection is to be established by checking through alleligibility criteria at screening and first baseline. A relevant record(e.g. checklist) of the eligibility criteria must be stored with thesource documentation at the study site. Deviation from any entrycriterion excludes a patient from enrollment into the study.

Inclusion Criteria (Both Parts)

-   Patients eligible for inclusion in this study have to fulfill all of    the following criteria:-   Written informed consent must be obtained before any assessment is    performed. If consent cannot be expressed in writing, it must be    formally documented and witnessed, ideally via an independent    trusted witness-   Age ≥18 years at screening.-   Hemodialysis-dependent for at least 2 months prior to screening.-   Receiving adequate hemodialysis at least 2 times per week for end    stage renal disease; adequate is defined as Kt/V≥1.2 at the most    recent monthly assessment prior to screening.-   Receiving chronic erythropoietin (EPO) therapy, as per the dialysis    site's anemia management protocol. EPO dose not increased by 50% or    more during 14 days prior to baseline. EPO therapy must be    short-acting formulation only (not darbepoetin) and administered IV    (not SC).-   Hgb ≥8.5, including Hgb ≥8.5 and <11.5 g/dL, and not increased by    ≥0.5 g/dL at baseline vs. prior 14 days.-   Ferritin <1500 ng/mL (inclusive) for at least 28 days prior to    baseline (may include screening). Alternatively, Ferritin >500    ng/mt: and ≤1000 ng/mL at screening.-   TSAT ≤30% at a minimum of one time point during the 90 days prior to    baseline, and TSAT ≤30% at baseline.

Exclusion Criteria (Both Parts)

-   Patients fulfilling, any of the following criteria are not eligible    for inclusion in this study. No additional exclusions may be applied    by the investigator, in order to ensure that the study population    will be representative of all eligible patients.-   1. Use of other investigational drugs within 5 half-lives of    enrollment, or until the expected pharmacodynamic effect has    returned to baseline, whichever is longer.-   2. History of hypersensitivity to the study drug or to therapeutic    antibodies.-   3. Known diagnosis of hemochromatosis.-   4. Known bone marrow malignancy, lymphatic malignancy or    myelodysplastic syndrome.-   5. History of dialysis AV fistula thrombosis within 2 months prior    to screening, or 2 or more episodes of AV fistula thrombosis within    6 months prior to screening.-   6. Severe co-morbid liver disease/dysfunction (Child-Pugh score ≥6)    or prior liver transplant 7. Heart failure (New York Heart    Association (NYHA) Functional Class III or IV)-   8. Gastrointestinal bleeding requiring intervention within the past    2 months of screening. Patients with Hepatitis C Virus (HCV)    infection may be included if all other liver function eligibility    criteria are met.-   9. ALT, AST or bilirubin ≥1.5×ULN within 4 weeks prior to baseline.-   10. Uncontrolled renal osteodystrophy defined as intact PTH≥750    pg/mL at screening.-   11. Conditions predisposing to an increased risk of serious    infection, such as an indwelling vascular catheter (central venous    line or hemodialysis catheter) or active infection requiring    antibiotic therapy at any time during the 2 weeks prior to    screening.-   12. Blood transfusion administered within 4 weeks prior to baseline.-   13. Receiving a loading dose (100 mg/week) IV iron within 1 week    prior to baseline.-   14. History of drug or alcohol abuse within the 12 months prior to    dosing, or evidence of such abuse as indicated by the laboratory    assays conducted during screening.-   15. A positive Hepatitis B surface antigen test result.-   16. History of immunodeficiency diseases, including a positive HIV    (ELISA and Western blot) test result.-   17. Women of childbearing potential may be enrolled in this study if    highly effective contraception is used, for a minimum of 125 days    following dosing with an antibody or antigen-binding fragment that    binds human BMP6. Highly effective contraception is defined as one    of the following: a. Total abstinence (when this is in line with the    preferred and usual lifestyle of the patient. Periodic abstinence    (e.g., calendar, ovulation, symptothermal, post-ovulation methods)    and withdrawal are not acceptable methods of contraception) b.    Male/female sterilization c. Use of oral, injected or implanted    hormonal methods of contraception or placement of an intrauterine    device (IUD) or intrauterine system (IUS) or other forms of hormonal    contraception that have comparable efficacy (failure rate <1%), for    example hormone vaginal ring or transdermal hormone contraception.

Treatment Investigational Treatment

The investigational therapy in this study is an antibody orantigen-binding fragment that binds human BMP6, for example an anti-BMP6IgG1, fully human antibody. The antibody is provided in liquid solution.The stock concentration will be diluted on site in accord with the doseto be administered. Infusion time will be maintained relatively constantacross cohorts at approximately 30 minutes. Part 1 will be open labelsingle dose, and Part 2 will be double-blinded, single dose, incomparison to a matching placebo (vehicle control). The anti-human BMP6Ab active substance and placebo will be supplied as liquid in vials. Theexcipients in the active and placebo are identical.

Treatment Arms

In Part 1, patients will be assigned to one of up to 6 dose cohortsconsisting of 6 patients each. Part 1 is an open label treatment. Thestarting dose, top dose, and dose adjustment rationale are describedabove. Provisional doses for Part 1 are given in Table 12 (Hgb <0.5 g/dLat MRSD) and Table 13 (Hgb ≥0.5 g/dL at MRSD).

TABLE 12 Provisional dose levels for Part 1 For Hgb less than 0.5Provisional Increment from g/dL at MRSD Dose level dose previous dose 1(MRSD) 0.010 mg/kg starting dose 2 0.016 mg/kg 60% ↑ 3 0.025 mg/kg 60% ↑4 0.040 mg/kg 60% ↑ 5 0.063 mg/kg 60% ↑ 6 (NOAEL) 0.100 mg/kg 60% ↑

-   This table is intended as an example of Part 1 dose adjustment for    guidance only. Intermediate or higher dose levels may be used and    some dose levels may be skipped based on data evaluation during the    informal interim analyses between each cohort. Actual dose levels    will be confirmed in writing by Novartis and provided to all    participating study sites before treatment of patients in a new    cohort.

TABLE 13 Provisional dose levels for Part 1 For Hgb greater than orequal to 0.5 g/dL at Provisional Increment from MRSD Dose level doseprevious dose 1 (MRSD) 0.0100 mg/kg starting dose 2 0.0010 mg/kg 90% ↓ 30.0016 mg/kg 60% ↑ 4 0.0025 mg/kg 60% ↑ 5 0.0040 mg/kg 60% ↑ 6 0.0063mg/kg 60% ↑

-   This table is intended as an example of Part 1 dose adjustment for    guidance only. Intermediate or higher dose levels may be used and    some dose levels may be skipped based on data evaluation during the    informal interim analyses between each cohort.-   Study treatments are defined as:-   A: single dose of placebo.-   B: single dose of anti-human BMP6 Ab at minimum PAD, as determined    in Part 1.-   C: single dose of anti-human BMP6 Ab at one dose level above minimum    PAD, as determined in Part 1.

Concomitant Treatment

All prescription medications, over-the-counter drugs and significantnon-drug therapies (including physical therapy and blood transfusions)administered or taken within the timeframe defined in the entry criteriaprior to the start of the study and during the study, must be recordedon the Concomitant medications/Significant non-drug therapies section ofthe CRF. Medication entries should be specific to trade name, the singledose and unit, the frequency and route of administration, the start anddiscontinuation date and the reason for therapy.

Efficacy/Pharmacodynamics

Efficacy assessments are specified below. Samples for efficacyassessments will be collected at various timepoints. Hematology labswill be assessed. Hgb and Fe indices will be reviewed during eachinter-cohort informal interim analysis as part of the dose adjustmentevaluation during Part 1 of the study. If the sample collection timesset initially are deemed suboptimal for understanding the relationshipbetween iron and PK, the sample collection times may be altered insubsequent cohorts in Part 1.

Iron Indices Panel

The anti-human BMP6 Ab is expected to mobilize Fe from body storesresulting in changes in serum Fe parameters including: serum Fe,transferrin saturation (TSAT), unbound Fe binding capacity (UIBC), totalFe binding capacity (TIBC), ferritin, and reticulocyte hemoglobincontent (CHr). These will be measured in serum using validated assays.

Safety

Safety assessments are specified below.

Physical Examination

A complete physical examination will include the examination of generalappearance, skin, neck (including thyroid), eyes, ears, nose, throat,lungs, heart, abdomen, back, lymph nodes, extremities, vascular andneurological. If indicated based on medical history and/or symptoms,rectal, external genitalia, breast, and/or pelvic exams may beperformed.

Significant findings that are present prior to the start of study drugmust be included in the Relevant Medical History/Current MedicalConditions screen on the patient's eCRF. Significant findings made afterthe start of study drug which meet the definition of an Adverse Eventmust be recorded on the Adverse Event screen of the patient's eCRF.

Vital Signs

-   Body temperature-   Blood pressure (BP)-   Pulse

Height and Weight

-   Height-   Body weight-   Body mass index (BMI) will be calculated (Body weight (kg)/[Height    (m)]2)

Laboratory Evaluations

Clinically relevant deviations of laboratory test results will beevaluated for criteria defining an adverse event and reported as such ifthe criteria are met. Repeated evaluations are mandatory untilnormalization of the result(s) or until the change is no longerclinically relevant.

Hematology

Hemoglobin, hematocrit, red blood cell count, white blood cell countwith differential and platelet count will be measured. Iron indices willbe monitored.

Clinical Chemistry

Sodium, potassium, creatinine, urea, chloride, albumin, calcium,alkaline phosphatase, total bilirubin, LDH, GGT, AST, and ALT will bemonitored. If the total bilirubin concentration is increased above 1.5times the upper limit of normal, direct and indirect reacting bilirubinshould be differentiated.

Electrocardiogram (ECG)

PR interval, QRS duration, heart rate, RR, QT, QTc

The Fridericia QT correction formula (QTcF) should be used for clinicaldecisions.

Pregnancy and Assessments of Fertility

Pregnancy tests are required of all female patients regardless ofreported reproductive/menopausal status.

Serum pregnancy tests will be performed for this study. If positive, thepatient must be discontinued from the trial.

When performed at screening and baseline, the result of this test mustbe received before the patient may be dosed,

Pharmacokinetics

PK samples will be collected. PK data will be reviewed during eachinter-cohort informal interim analysis as part of the dose adjustmentevaluation during Part 1 of the study. If the sample collection timesset initially are deemed inadequate or inappropriate for characterizingthe PK profile, the sample collection times may be altered in subsequentcohorts. The number of blood draws and total blood volume collected willnot exceed those stated in the protocol.

PK samples will be collected and evaluated in all patients at all doselevels.

The concentration of free anti-human BMP6 Ab will be determined using anELISA assay. The anticipated lower limit of quantification (LLOQ) is 10pg/mL.

Untreated (placebo) samples will not be analyzed.

Free anti-human BMP6 Ab concentrations will be expressed at μg/mL. Allconcentrations below the LLOQ or missing data will be labeled as such inthe concentration data listings. Concentrations below the LLOQ will betreated as zero in summary statistics for concentration data only. Theywill not be considered in the calculation of PK parameters.

PK samples remaining after determination of free anti-human BMP6 Ab maybe used for exploratory assessments or other bioanalytical purposes(e.g. cross-check between different sites, stability assessment).

The following pharmacokinetic parameters will be determined (iffeasible) using non-compartmental method(s) with Phoenix WinNonlin(Version 6.2 or higher): Cmax, tmax, AUC(0-t), AUC(0-tlast), Cmax/D, andAUC/D based on the serum concentration-time data. The linear trapezoidalrule will be used for AUC calculations. The terminal half-life of anantibody or antigen-binding fragment that binds human BMP6 (t_(1/2))will also be estimated if feasible based on the data.

Other Assessments Immunogenicity

An ELISA assay will be used to detect anti-human BMP6 antibodies. IGsamples remaining after immunogenicity analysis may be used forexploratory assessment or other bioanalytical purposes (e.g.,cross-check between different sites).

Exploratory Assessments

Biomarkers are objectively measured and evaluated indicators of normalbiological processes, pathogenic processes, or pharmacologic responsesto a therapeutic intervention (Biomarkers Definitions Working Group2001).

The BMP6-hepcidin pathway is as follows: BMP6 signalling in hepatocytesis required for induced expression of hepcidin, inhibiting enterocyteiron absorption and macrophage iron export. BMP6-neutralizing antibodyas a hepcidin-lowering therapy should benefit patients withiron-restricted anemia by reducing EPO requirement and increasing thenumber of patients who reach target Hgb level.

Based on the above described biology, exploratory biomarker assessmentsinclude, but not limited to hepcidin (measured using LC-MS assay).

Additional exploratory assessments may investigate potential roles ofbone absorption markers, as well as address inflammation as a factorcontributing to the mechanism of action.

-   The exploratory objectives are as follows:-   To assess the relationships between hepcidin levels and several key    measures such as ERI and iron indices:-   To study the dynamics between primary and secondary endpoints and    exploratory biomarkers longitudinally;-   To assess pharmacogenetics;-   To assess immunogenicity-   Sample(s) will be collected at various time point(s).-   Further details on sample collection, numbering, processing and    shipment will be provided in a central lab manual.

DNA

Exploratory DNA research studies are planned as a part of this study theobjectives of identifying genetic factors which may (1) be related toerythropoietin-treated chronic hemodialysis patients with functionaliron-deficiency anemia, (2) predict response to treatment withanti-human BMP6 Ab, or (3) predict genetic predisposition to sideeffects.

In addition, recent advances in genotyping technologies have madegenome-wide approaches possible. Genome-wide approaches may also beundertaken within the restricted scope of these studies as describedabove.

Soluble Biomarkers

Hepcidin will be quantified in plasma as a potential PD/biomarker.

Detailed descriptions of the assays will be included in thebioanalytical data reports.

Other Biomarkers

Hypothesis-free platforms might be used to understand diseaseheterogeneity, mode of action and/or potential identification ofstratification markers. Immunogenicity (IG) samples will be collected atvarious timepoints. Immunogenicity of anti-human BMP6 Ab will beassessed by measuring antibodies recognizing the anti-human BMP6antibody.

REFERENCES

-   Fukuma S, Yamaguchi T, Hashimoto S et al (2012)    Erythropoiesis-stimulating agent responsiveness and mortality in    hemodialysis patients: results from a cohort study from the dialysis    registry in Japan. Am J Kidney Dis; 59(1): p. 108-16.-   Kilpatrick R D, Critchlow C W, Fishbane S et al (2008) Greater    epoetin alfa responsiveness is associated with improved survival in    hemodialysis patients. Clin J Am Soc Nephrol; 2008. 3(4):1077-83.-   Lopez-Gomez J M, Portoles T M, and Aljama P (2008), Factors that    condition the response to erythropoietin in patients on hemodialysis    and their relation to mortality. Kidney Int Suppl; (111):S75-81.-   Meynard D, Kautz L, Darnaud V et al (2009) Lack of the bone    morphogenetic protein BMP6 induces massive iron overload. Nat Genet;    41(4):478-81.-   Salim S, Patenaude V, Abenhaim H A (2015) Pregnancy outcomes among    renal transplant recipients and patients with end-stage renal    disease on dialysis. J Perinat Med (Epub ahead of print)    http://www.ncbi.nlm.nih.gov/pubmed/25719292.-   Suttorp M M, Hoekstra T, Rotmans J I et al (2013)    Erythropoiesis-stimulating agent resistance and mortality in    hemodialysis and peritoneal dialysis patients. BMC Nephrol;    14(1):200.-   Zaritsk J, Young B, Gales B, et al (2010) Reduction of serum    hepcidin by hemodialysis in pediatric and adult patients. Clin J Am    Soc Nephrol; 5(6):1010-14.

Example 3 TSAT Levels for Patients Treated with 0.01 mg/kg Antibody 7

Data, including TSAT (iron saturation, %) levels were assessed for thefirst 10 patients treated according to the clinical protocol describedin Example 2, with each patient receiving a single infusion of 0.01mg/kg Antibody 7. None of these patients demonstrated any liver safetysignals that defined the no observed adverse effect level (NOAEL) of 0.1mg/kg/week. Cohort 1 included 5 anemic hemodialysis patients with lowferritin levels of less than or equal to 500 ng/mL, while Cohort 2included 5 anemic hemodialysis patients higher ferritin levels (between500 and 1000 ng/mL).

In the five Cohort 1 (low ferritin) patients who received 0.01 mg/kgAntibody 7, post-dose TSAT levels increased by an average of only 9.8%(mean 38.6% post-dose vs. 24.8% pre-dose). In contrast, post-dose TSATlevels increased by an average of 17.6% (mean 48.4% post-dose vs. 30.8%pre-dose) in five Cohort 2 (high ferritin) patients who received 0.01mg/kg Antibody 7. The data are shown in FIG. 14, which shows the peakTSAT levels pre-Antibody 7 vs. within 72 hours post-Antibody 7administration in the 2 cohorts. Surprisingly, in contrast to the Cohort1 patients, the Cohort 2 anemia patients demonstrate a distinct TSATincrease in comparison to baseline. These data indicate that patientswith ferritin levels greater than or equal to 500 ng/mL are goodcandidates for response to anti-BMP6 therapy, and that ferritin level,for example, a ferritin level greater than or equal to 500 ng/mL, may bean indicator of response.

Unless defined otherwise, the technical and scientific terms used hereinhave the same meaning as that usually understood by a specialistfamiliar with the field to which the disclosure belongs.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein. Unless indicated otherwise,each of the references cited herein is incorporated in its entirety byreference.

Claims to the invention are non-limiting and are provided below.

Although particular aspects and claims have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims, or the scope of subject matter ofclaims of any corresponding future application. In particular, it iscontemplated by the inventors that various substitutions, alterations,and modifications may be made to the disclosure without departing fromthe spirit and scope of the disclosure as defined by the claims. Thechoice of nucleic acid starting material, clone of interest, or librarytype is believed to be a matter of routine for a person of ordinaryskill in the art with knowledge of the aspects described herein. Otheraspects, advantages, and modifications considered to be within the scopeof the following claims. Those skilled in the art will recognize or beable to ascertain, using no more than routine experimentation, manyequivalents of the specific aspects of the invention described herein.Such equivalents are intended to be encompassed by the following claims.Redrafting of claim scope in later filed corresponding applications maybe due to limitations by the patent laws of various countries and shouldnot be interpreted as giving up subject matter of the claims.

What is claimed is:
 1. A method of selectively: a. inhibiting BMP6; b.increasing serum iron levels, transferrin saturation (TAST),reticulocyte hemoglobin content (CHr), reticulocyte count, red bloodcell count, hemoglobin, or hematocrit; c. reducing the activity or levelof Hepcidin; d. treating anemia; or e. increasing or maintaininghemoglobin level; in a patient in need thereof, comprising selectivelyadministering a therapeutically effective amount of a BMP6 antagonist tothe patient on the basis of a biological sample from the patient havinga ferritin level of ≤2000 ng/mL. 2.-4. (canceled)
 5. A method ofselectively: a. inhibiting BMP6; b. increasing serum iron levels,transferrin saturation (TAST), reticulocyte hemoglobin content (CHr),reticulocyte count, red blood cell count, hemoglobin, or hematocrit; c.reducing the activity or level of Hepcidin; d. treating anemia; or e.increasing or maintaining hemoglobin level; in a patient in needthereof, comprising selectively administering a therapeuticallyeffective amount of a BMP6 antagonist to the patient on the basis of abiological sample from the patient having a ferritin level of ≥500ng/mL. 6.-19. (canceled)
 20. The method of claim 1, wherein the ferritinlevel is ferritin protein level. 21.-23. (canceled)
 24. The method ofclaim 1, wherein the anemia is anemia associated with chronic disease.25. The method of claim 24, wherein the chronic disease is chronickidney disease, cancer or inflammation.
 26. The method of claim 1,wherein the patient is being or has been treated with an erythropoiesisstimulating agent (ESA).
 27. The method of claim 26, wherein the ESA iserythropoietin (EPO).
 28. The method of claim 1, wherein the anemia isEPO-hyporesponsive anemia.
 29. The method of claim 1, wherein the anemiais iron restricted anemia, e.g., functional iron-restricted anemia. 30.The method of claim 1, wherein the patient is a chronic hemodialysispatient.
 31. The method of claim 1, further comprising reducing thepatient's iron dose requirement, reducing the patient's EPO doserequirement, or reducing both the patient's iron dose requirement andthe patient's EPO dose requirement, relative to said EPO doserequirement and/or iron dose requirement in the absence of treatmentwith the therapeutically effective amount of the BMP6 antagonist.32.-35. (canceled)
 36. The method of claim 35, wherein the BMP6antagonist is an anti-BMP6 antibody or antigen-binding fragment thereof.37. (canceled)
 38. The method of claim 36, wherein the anti-BMP6antibody or antigen-binding fragment thereof comprises: (a) HCDR1,HCDR2, and HCDR3 sequences of SEQ ID NOs: 69, 70 and 71, respectively,and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 79, 80 and 81,respectively; (b) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 72,73 and 74, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ IDNOs: 82, 83 and 84, respectively; (c) HCDR1, HCDR2, and HCDR3 sequencesof SEQ ID NOs: 29, 30 and 31, respectively, and LCDR1, LCDR2, and LCDR3sequences of SEQ ID NOs: 39, 40 and 41, respectively; (d) HCDR1, HCDR2,and HCDR3 sequences of SEQ ID NOs: 32, 33 and 34, respectively, andLCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 42, 43 and 44,respectively; (e) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 49,50 and 51, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ IDNOs: 59, 60 and 61, respectively; (f) HCDR1, HCDR2, and HCDR3 sequencesof SEQ ID NOs: 52, 53 and 54, respectively, and LCDR1, LCDR2, and LCDR3sequences of SEQ ID NOs: 62, 63 and 64, respectively; (g) HCDR1, HCDR2,and HCDR3 sequences of SEQ ID NOs: 9, 10 and 11, respectively, andLCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 19, 20 and 21,respectively; or (h) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:12, 13 and 14, respectively, and LCDR1, LCDR2, and LCDR3 sequences ofSEQ ID NOs: 22, 23 and 24, respectively.
 39. The method of claim 36,wherein the anti-BMP6 antibody or antigen-binding fragment thereofcomprises: (a) A VH sequence of SEQ ID NO: 75; (b) A VH sequence of SEQID NO: 35; (c) A VH sequence of SEQ ID NO: 55; or (d) A VH sequence ofSEQ ID NO:
 15. 40. The method of claim 36, wherein the anti-BMP6antibody or antigen-binding fragment thereof comprises: (a) A VLsequence of SEQ ID NO: 85; (b) A VL sequence of SEQ ID NO: 45; (c) A VLsequence of SEQ ID NO: 65; or (d) A VL sequence of SEQ ID NO:
 25. 41.The method of claim 36, wherein the anti-BMP6 antibody orantigen-binding fragment thereof comprises: (a) A VH sequence of SEQ IDNO: 75; and a VL sequence of SEQ ID NO: 85; (b) A VH sequence of SEQ IDNO: 35; and a VL sequence of SEQ ID NO: 45; (c) A VH sequence of SEQ IDNO: 55; and a VL sequence of SEQ ID NO: 65; or (d) A VH sequence of SEQID NO: 15; and a VL sequence of SEQ ID NO:
 25. 42. The method of claim36, wherein the anti-BMP6 antibody or antigen-binding fragment thereofcomprises: (a) A heavy chain sequence of SEQ ID NO: 77; (b) A heavychain sequence of SEQ ID NO: 37; (c) A heavy chain sequence of SEQ IDNO: 57; or (d) A heavy chain sequence of SEQ ID NO:
 17. 43. The methodof claim 36, wherein the anti-BMP6 antibody or antigen-binding fragmentthereof comprises: (a) A light chain sequence of SEQ ID NO: 87; (b) Alight chain sequence of SEQ ID NO: 47; (c) A light chain sequence of SEQID NO: 67; or (d) A light chain sequence of SEQ ID NO:
 27. 44. Themethod of claim 36, wherein the anti-BMP6 antibody or antigen-bindingfragment thereof comprises: (a) A heavy chain sequence of SEQ ID NO: 77;and a light chain sequence of SEQ ID NO: 87; (b) A heavy chain sequenceof SEQ ID NO: 37; and a light chain sequence of SEQ ID NO: 47; (c) Aheavy chain sequence of SEQ ID NO: 57; and a light chain sequence of SEQID NO: 67; or (d) A heavy chain sequence of SEQ ID NO: 17; and a lightchain sequence of SEQ ID NO:
 27. 45. The method of claim 36, wherein theanti-BMP6 antibody or antigen-binding fragment thereof: a) binds humanBMP6 with a KD of ≤1 nM; or b) binds human BMP6 with a KD of ≤0.1 nM.46. The method of claim 36, wherein the anti-BMP6 antibody orantigen-binding fragment thereof: a) has at least about 100-fold greateraffinity for human BMP6 than human BMP7; b) has at least about 100-foldgreater affinity for human BMP6 than human BMP2, human BMPS, or humanBMP7; c) has at least about 500-fold greater affinity for human BMP6than human BMP2, human BMPS, or human BMP7; and/or d) has no detectablebinding to human BMP2 and/or BMP7 in an ELISA.
 47. (canceled)
 48. Themethod of claim 36, wherein the anti-BMP6 antibody or antigen-bindingfragment thereof is an IgG selected from an IgG1, an IgG2, and IgG3 oran IgG4.
 49. The method of claim 36, wherein the anti-BMP6 antibody orantigen-binding fragment thereof is selected from the group consistingof: a monoclonal antibody, a chimeric antibody, a single chain antibody,a Fab and a scFv.
 50. The method of claim 36, wherein the anti-BMP6antibody or antigen-binding fragment thereof is a component of animmunoconjugate.
 51. The method of claim 36, wherein the anti-BMP6antibody or antigen-binding fragment thereof has altered effectorfunction through mutation of the Fc region.
 52. The method of claim 36,wherein the anti-BMP6 antibody or antigen-binding fragment thereof bindsto a human BMP6 epitope comprising the sequence QTLVHLMNPEYVPKP (SEQ IDNO: 98). 53.-56. (canceled)
 57. The method of claim 36, wherein theantibody or antigen-binding fragment thereof is administered: a)intravenously; or b) subcutaneously. 58.-63. (canceled)